Screening methods for biologically active ligands

ABSTRACT

In the case when there are two objective biological activities, and the aim is to isolate a compound having at least one biological activity, the present inventors developed an assay method wherein a common detection marker is utilized for separately detecting the presence or absence of each of the biological activities. The present inventors discovered that a compound having at least one of two or more distinct biological activities can be efficiently and conveniently detected by simultaneously assaying at least one test sample or more by the above-mentioned method. Furthermore, for a test sample that proved to be positive by the detection method, they found that it is possible to efficiently and conveniently screen for a test sample having an objective specific biological activity by combining with a method wherein an individual activity of a test sample can be detected to specify the biological activity.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a national phase application under 35 U.S.C.Section 371 filed from International Patent Application PCT/JP01/06170,filed 17 Jul. 2001, which claims priority to Japanese patent applicationSerial. No. JP 2000-221070, filed 17 Jul. 2000 and to Japanese patentapplication Serial. No. JP 2001-159032, filed May 28, 2001. The contentsof these applications are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

This invention relates to efficient screening methods for biologicallyactive substances that bind receptors.

BACKGROUND ART

Hematopoietic factors represented by erythropoietin (EPO) andgranulocyte-colony stimulating factor (G-CSF) have already beendeveloped as pharmaceutical agents, and have been utilized for thetreatment of various diseases. Cytokines, such as interferon, andhormones, including insulin and growth hormone, are also commerciallyavailable as pharmaceutical agents. Most of the biologically activeproteins used as pharmaceutical agents have been produced by geneticengineering techniques. Low molecular weight compounds are beingscreened as the next-generation drugs replacing biologically activeproteins. For instance, if it was possible to find low molecular weightcompounds having the same activities as biologically active proteins,such compounds will be useful as pharmaceutical agents, since they maybe orally administered. Therefore, there is a need to develop efficientmethods for screening low molecular weight compounds that havebiological activities similar to naturally existing ligands.

Several screening methods are already known. For example, when screeningfor a ligand of a receptor whose amino acid sequence and function areknown, there is a method in which a chimeric receptor comprising theintracellular domain of a receptor with a known function and theextracellular domain of a receptor of interest is prepared, and thechimeric protein is used to screen for a ligand of the receptor ofinterest (Ishizaka-Ikeda, E., Pro. Natl. Acad. Sci. USA (1993) 90,p123-127; U.S. Pat. No. 4,859,609 and U.S. Pat. No. 5,030,576). Usingthe insulin receptor as the extracellular domain and the EGF receptor asthe intracellular domain, these US patents describe a method that assaysin a cell-free system chimeric receptor phosphorylation induced by thebinding of a ligand. A method using a chimeric receptor that consists ofthe extracellular domain of the EGF receptor and the intracellulardomain of the EPO receptor, has also been reported (WO94-29458). Theseapproaches may also be applicable to the screening of ligands for anorphan receptor, whose natural ligand is not yet known.

However, in each of these screening methods, only one kind of receptoris used as the receptor of interest, and one screening can only detectthe effect a test sample has on that single receptor. Therefore, inorder to assay the effect on several receptors, the same number ofscreenings as that of receptors of interest is required. Also, theseapproaches are inefficient for a preliminary screening of a vast numberof test samples that have different structures and whose activities areunconfirmed, since activities of most ligands cannot be detected. It is,therefore, necessary to develop a method that can efficiently andrapidly screen a vast number of test samples.

DISCLOSURE OF THE INVENTION

The present invention provides an efficient method of screening forsubstances that bind biologically active receptors.

In the case when there are two or more objective biological activities,and the aim is to isolate a compound having at least one of thebiological activities, exhaustive research conducted by the presentinventors led to the development of an assay method wherein a commondetection marker is utilized for separately detecting the presence orabsence of each of the biological activities. The present inventorscompleted the invention by discovering that a compound having at leastone of two or more distinct biological activities can be efficiently andconveniently detected by simultaneously assaying at least one testsample or more by the above-mentioned method. Furthermore, for a testsample that proved to be positive by the detection method, they foundthat it is possible to efficiently and conveniently screen for a testsample having an objective specific biological activity by combiningwith a method wherein an individual activity of a test sample can bedetected to specify the biological activity.

Specifically, the present invention provides:

-   (1) a method for simultaneously detecting two or more distinct    activities, wherein said method comprises the steps of:    -   (i) determining in advance, a detection marker common to said        activities,    -   (ii) preparing a detection method that can detect each of said        two or more distinct activities using said common detection        marker, and    -   (iii) simultaneously detecting said activities using said        detection method;-   (2) a method for selecting a test sample that has at least one of    two or more distinct activities, wherein said method comprises the    steps of:    -   (i) determining in advance, a detection marker common to said        activities,    -   (ii) preparing a detection method that can detect each of said        two or more distinct biological activities using said common        detection marker, and    -   (iii) simultaneously detecting at least one of the biological        activities of a test sample using said detection method;-   (3) a method of screening for a ligand that can bind to at least one    of two or more kinds of receptors, wherein said method comprises the    steps of:    -   (i) obtaining two or more kinds of receptors comprising (a) a        common signal-transducing domain, and (b) a domain other than        the signal-transducing domain, wherein said domain derives from        the same receptor from which the common domain of (a) is derived        and/or a different receptor,    -   (ii) contacting a test sample with said two or more kinds of        receptors, and    -   (iii) detecting the biological activity of said test sample;-   (4) the method according to (3), wherein said step (i) obtains a    cell expressing two or more kinds of receptors comprising (a) a    common signal-transducing domain, and (b) a domain other than the    signal-transducing domain, wherein said domain derives from the same    receptor from which the common domain of (a) is derived and/or a    different receptor;-   (5) the method according to (3), wherein said step (i) obtains two    or more kinds of cells that express a receptor comprising (a) a    common signal-transducing domain, and (b) a domain other than the    signal-transducing domain, wherein said domain derives from the same    receptor from which the common domain of (a) is derived and/or a    different receptor;-   (6) a method according to any one of (3) to (5), wherein said    signal-transducing domain and/or said domain other than the    signal-transducing domain is derived from a cell membrane receptor;-   (7) a method according to any one of (3) to (5), wherein said    signal-transducing domain and/or said domain other than the    signal-transducing domain is derived from a nuclear receptor;-   (8) the method according to (6), wherein said domain other than the    signal-transducing domain is the extracellular domain, of a cell    membrane receptor, or a portion thereof;-   (9) the method according to (6), wherein said domain other than the    signal-transducing domain is a ligand-binding domain of a cell    membrane receptor;-   (10) the method according to (6), wherein said signal-transducing    domain and/or said domain other than the signal-transducing domain    is derived from a receptor belonging to a receptor family selected    from the group consisting of the hematopoietic factor receptor    family, cytokine receptor family, tyrosine kinase-type receptor    family, serine/threonine kinase-type receptor family, TNF receptor    family, G protein-coupled receptor family, GPI-anchored receptor    family, tyrosine phosphatase-type receptor family, cell adhesion    receptor family, and hormone receptor family;-   (11) the method according to (6), wherein said signal-transducing    domain and/or said domain other than the signal-transducing domain    is derived from the following receptors: human or mouse    erythropoietin (EPO) receptor, human or mouse granulocyte-colony    stimulating factor (G-CSF) receptor, human or mouse thrombopoietin    (TPO) receptor, human or mouse receptor, human or mouse Flt-3, human    or mouse platelet-derived growth factor (PDGF) receptor, human or    mouse interferon (IFN)-α or -β receptor, human or mouse leptin    receptor, human or mouse growth hormone (GH) receptor, human or    mouse interleukin (IL)-10 receptor, human or mouse insulin-like    growth factor (IGF)-I receptor, human or mouse leukemia inhibitory    factor (LIF) receptor, or human or mouse ciliary neurotrophic factor    (CNTF) receptor;-   (12) the method according to (11), wherein said signal-transducing    domain derives from mouse G-CSF receptor;-   (13) the method according to (3), wherein in step (ii), two or more    kinds of cells expressing said receptors are mixed and contacted    with the test sample;-   (14) the method according to any one of (4) to (13), wherein said    cell(s) is a transformed cell(s);-   (15) the method according to (14), wherein said cell(s) is derived    from a cytokine-dependent cell;-   (16) the method according to (15), wherein said transformed cell(s)    is derived from Ba/F3 cells or FDC-P1 cells;-   (17) a method according to (2) or (3), wherein in step (iii), an    agonist or antagonist activity of the test sample is detected;-   (18) the method according to (2) or (3), wherein in step (iii), the    biological activity of the test sample is measured by using a    cell-free detection system;-   (19) the method according to (2) or (3), wherein in step (iii), the    biological activity of the test sample is measured by using a    cell-based detection system;-   (20) the method according to (19), wherein in step (iii), the    biological activity of the test sample is measured using a    phenotypic change in the cell;-   (21) the method according to (20), wherein said phenotypic change is    a quantitative and/or qualitative change of a cell surface antigen;-   (22) the method according to (20), wherein said phenotypic change is    a change in proliferation activity;-   (23) the method according to (3), which method further comprises the    step of measuring the biological activity of a test sample by    contacting it with one of said two or more receptors obtained in (i)    in order to determine the specificity of the test sample towards the    receptor;-   (24) a cell that expresses two or more kinds of receptors    comprising (a) a common signal-transducing domain, and (b) a domain    other than the signal-transducing domain, wherein said domain    derives from the same receptor from which the common domain of (a)    is derived and/or a different receptor;-   (25) a kit for screening a substance or ligand that binds to at    least one of two or more kinds of receptors, wherein said kit    comprises one of (a) to (c):-   (a) two or more kinds of receptors comprising (a) a common    signal-transducing domain, and (b) a domain other than the    signal-transducing domain, wherein said domain derives from the same    receptor from which the common domain of (a) is derived and/or a    different receptor;-   (b) DNA encoding two or more kinds of receptors comprising (a) a    common signal-transducing domain, and (b) a domain other than the    signal-transducing domain, wherein said domain derives from the same    receptor from which the common domain of (a) is derived and/or a    different receptor;-   (c) a cell expressing two or more kinds of receptors comprising (a)    a common signal-transducing domain, and (b) a domain other than the    signal-transducing domain, wherein said domain derives from the same    receptor from which the common domain of (a) is derived and/or a    different receptor;-   (26) a substance or ligand isolated by the screening according    to (2) or (3); and-   (27) a pharmaceutical composition comprising the substance or ligand    according to (26).

The present invention converted a method that detected activities usingdifferent markers into a test method that detects activities using apredetermined detection marker. Thereby, the present invention developeda method that simultaneously detects two or more distinct activitieshaving a common detection marker. The two or more activities are notrestricted to any particular activities if the activities includeseveral, distinct activities, but are preferably biological activities.“Biological activities” mean activities that can influence or causequantitative and/or qualitative changes in a living body, tissue, cell,protein, DNA, RNA, etc. The two or more different biological activitiescan be any combinations of any biological activities if the activitiescan be detected by using a common detection marker. Biologicalactivities include, cytokine activity, enzyme activity, transcriptionactivity, membrane transport activity, and binding activity, etc.Examples of enzyme activities are, proteolytic activity,phosphorylation/dephosphorylation activity, redox activity, transferactivity, nucleic acid degradation activity, and dehydration activity.Furthermore, antigen-antibody reaction, and binding and/or activation ofcell adhesion factors are examples of binding activities. It ispreferable to use two or more biological activities of the samecategory, since it is relatively easy to design a detection method usinga common marker.

The two or more activities can be measured by using different markersfor each of the activities. However, the predetermined detection markerused in the present invention, is one of the distinct markers used toassay the two or more activities, or is a marker that is different fromthose markers. As detection markers used for the methods of the presentinvention, any markers can be utilized if quantitative and/orqualitative changes can be measured. For example, markers for cell-freeor cell-based assays, tissue-specific markers, and organism-specificmarkers can be used. As markers for cell-free systems, enzyme reactionsand quantitative and/or qualitative changes in proteins, DNA and RNA canbe used. As an enzyme reaction, for instance, an amino acid transferreaction, a glycosyl transfer reaction, a dehydration reaction, adehydrogenation reaction, and a substrate-cleaving reaction can be used.Furthermore, protein phosphorylation, dephosphorylation, dimerization,multimerization, degradation, and dissociation, and such, and DNA or RNAamplification, digestion and elongation can also be used. For example,downstream protein phosphorylation in signal transduction pathways canbe used as a detection marker. As markers for cell-based systems,changes in cellular phenotype, for example, quantitative and/orqualitative changes-in cellular products, cell proliferation activitychanges, morphological changes, changes in cellular characteristics, andsuch can be used. As cellular products, secretory proteins, cell-surfaceantigens, intracellular proteins, mRNA, and such can be used. Ascellular morphological changes, changes in: the formation and/or numberof cellular protrusions; the degree of cell flatness; cellularelongation or the ratio between cellular length and width; cell-size;intracellular structure; heterogeneity or homogeneity in the cellpopulation; cell density; and such can be used. These morphologicalchanges can be recognized by microscopic observations. As changes incellular characteristics, changes in anchorage dependency, cytokinedependency, hormone dependency, drug resistance, cell motility, cellmigration activity, pulsatility, intracellular substance, and such canbe used. Cell motility can be assayed by measuring cell invasion andmigration activities. As changes in intracellular substances, enzymeactivities, mRNA quantity, amounts of intracellular signal transducerssuch as Ca²⁺ and cAMP, protein contents, and such can be used. To selecta compound having an agonist activity for a cell-membrane receptor, achange in cell growth activity induced by the stimulation of thereceptor can be used as a marker. For tissues, functional changes inrespective tissues can be used as detection markers. As markers forliving organisms, changes in tissue weight, changes in the blood system,for example, changes in blood cell counts, protein contents, enzymeactivities, electrolyte amounts; as well as changes in the circulatorysystem (for example, changes in blood pressure and heart rate, etc.) canbe used.

These detection markers are not restricted, and luminescence, coloring,fluorescence, radio activity, fluorescence polarity, surface plasmonresonance signal, time-resolved fluorescence, mass, absorption spectra,light scattering, and fluorescence resonance energy transfer, and suchcan be used. These methods are well known to those skilled in the art,and one can select an appropriate method for one's purpose. For example,absorption spectra, luminescence, and fluorescence can be measured byusing generally used photometers and plate readers, luminometers, andfluorometers, respectively. Mass can be measured by using a massspectrometer. Radiation can be measured by using an instrument such as agamma counter depending on the type of radioactive ray; fluorescencepolarity can be measured by using BEACON (Takara Shuzo Co., Ltd);surface plasmon resonance signals can be measured by using BIACORE;time-resolved fluorescence, fluorescence resonance energy transfer, andsuch can be measured by using ARVO, etc. Flow cytometers and such canalso be used for the measurement. Each of these methods can be used toassay two or more detection markers, and may also be used for thesimultaneous and/or continuous assay of two or more different markers ifsuch an assay would be convenient. For instance, a fluorometer cansimultaneously measure fluorescence and fluorescence resonance energytransfer.

One embodiment of the methods according to the present invention is ascreening method for the selection of compounds having at least one oftwo or more distinct biological activities. For example, the method canbe used for the selection of a ligand that can bind one of two or moredifferent receptors of interest.

In the present invention, “ligand” means a substance that has anactivity to bind a receptor, and that is able to induce biologicalactivities through the binding to the receptor. Among the ligands, asubstance that is produced by a living organism and that has abiological activity in an organism is called a natural ligand.

In the methods of the present invention, any sample whose biologicalactivity is to be detected can be used as a test sample. Examples oftest samples are cell extracts, cell culture supernatants, microorganismfermentation products, marine organism extracts, plant extracts,purified or crude proteins, peptides, non-peptide compounds, syntheticlow-molecular-weight compounds, and natural compounds; but are notlimited to these examples.

Any kind of receptor molecule can be used in the methods of the presentinvention, if upon ligand binding, the receptor can induce changes in acharacteristic of a detection marker. For example, cell membranereceptors, nuclear receptors, and intracellular receptors can be used.Cell membrane receptors are receptors expressed on the cell surface, andupon ligand binding to the extracellular domain of the receptor, cantransmit a signal into the cell and induce some biological change.Specifically, receptor molecules belonging to the following families canbe used; the hematopoietic factor receptor family, cytokine receptorfamily, tyrosine kinase-type receptor family, serine/threoninekinase-type receptor family, TNF receptor family, G protein-coupledreceptor family, GPI-anchored receptor family, tyrosine phosphatase-typereceptor family, cell adhesion receptor family, and hormone receptorfamily, etc.

Characteristics of the receptors of these families can be found invarious literatures, for example, in the following: Cooke BA., KingRJB., van der Molen HJ. ed. New Comprehesive Biochemistry Vol.18B“Hormones and their Actions Part II” pp.1-46 (1988) Elsevier SciencePublishers BV., New York, USA; Patthy L. (1990) Cell, 61: 13-14.;Ullrich A., et al. (1990) Cell, 61: 203-212.; Massagul J. (1992) Cell,69: 1067-1070.; Miyajima A., et al. (1992) Annu. Rev. Immunol., 10:295-331.; Taga T. and Kishimoto T. (1992) FASEB J., 7: 3387-3396.; FantlWI., et al. (1993) Annu. Rev. Biochem., 62: 453-481.; Smith CA., et al.(1994) Cell, 76: 959-962.; Flower DR. (1999) Biochim. Biophys. Acta,1422: 207-234.; Miyasaka M. ed. Cell Technology, Handbook Series“Handbook for adhesion factors” (1994) Shujunsha, Tokyo, Japan, etc. Asspecific receptors belonging to the families, the following moleculescan preferably be used in the present invention: for example, human ormouse erythropoietin (EPO) receptor, human or mouse granulocyte-colonystimulating factor (G-CSF) receptor, human or mouse thrombopoietin (TPO)receptor, human or mouse insulin receptor, human or mouse Flt-3 ligandreceptor, human or mouse platelet-derived growth factor (PDGF) receptor,human or mouse interferon (rFN)-α or -β receptor, human or mouse leptinreceptor, human or mouse growth hormone (GH) receptor, human or mouseinterleukin (IL)-10 receptor, human or mouse insulin-like growth factor(IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF)receptor, and human or mouse ciliary neurotrophic factor (CNTF)receptor. Sequences of these receptors are well known (hEPOR: Jones, SS.et al. (1990) Blood, 76, 31-35; mEPOR: D'Andrea, AD. et al. (1989) Cell57, 277-285.; hG-CSFR: Fukunaga, R. et al. (1990) Proc. Natl. Acad. Sci.USA. 87, 8702-8706.; mG-CSFR: Fukunaga, R. et al. (1990) Cell 61,341-350.; hTPOR: Vigon, I. et al. (1992) 89, 5640-5644.; mTPOR: Skoda,RC. et al. (1993) 12, 2645-2653.; hInsR: Ullrich, A. et al. (1985)Nature 313, 756-761.; hFlt-3: Small, D. et al. (1994) Proc. Natl. Acad.Sci. USA. 91, 459-463.; hPDGFR: Gronwald, RGK. et al. (1988) Proc. Natl.Acad. Sci. USA. 85, 3435-3439.; hIFNα/β R: Uze, G. et al. (1990) Cell60, 225-234.; and Novick, D. et al. (1994) Cell 77, 391-400). Nuclearreceptors are receptors that can bind specific DNA sequences followingligand binding, and regulate the transcription activity of mRNA.Examples are, the steroid and retinoid X receptor families, and such.The steroid receptor family includes the glucocorticoid receptor,mineral corticoid receptor, progesterone receptor, androgen receptor,and estrogen receptor. The retinoid X receptor family includes retinoicacid receptor, thyroid hormone receptor, and vitamin D3 receptor.Intracellular receptors are receptors that exist inside the cell, andinduce biological activities in the cell, upon binding to variousligands.

In a method for selecting a substance or ligand that can bind any one oftwo or more receptors of interest, a common functional domain of thereceptors can be used for developing a method for detecting the commonmarker. A functional domain is a receptor domain required for theinduction of a biological activity, and is usually a domain distinctfrom the ligand-binding domain. When receptors are cell membranereceptors capable of binding to secretory proteins such as cytokines orhematopoietic factors, an intracellular domain of the receptor,preferably the signal-transducing domain, can be used as the functionaldomain. Therefore, a method utilizing a common detection marker can bedeveloped by constructing chimeric receptors having a common signalingdomain with the same amino acid sequence, and different extracellulardomains. As a marker for cell-free systems, the formation of multimers,preferably dimers, of the receptors can be used. In the case of cytokinereceptors, it is known that signal transduction is induced byligand-induced dimerization or multimerization of the receptors. Hence,receptor dimerization or multimerization can be used as a detectionmarker. For example, detection can be done by directly immobilizing areceptor onto an immunoplate, or by immobilizing a biotinylated receptoronto an avidin-immobilized plate, mixing a test sample and aradio-labeled receptor, and detecting a test sample that promotes thebinding of the radio-labeled receptor to the immobilized receptor byscintillitant proximal assay (SPA) (WO99-53313; or Qureshi, S A, et al.,Proc. Natl. Acad. Sci. USA, (1999) 96, p12156-12161). Phosphorylation,dephosphorylation, degradation, and such of receptor molecules can alsobe appropriately used as detection markers. Phosphorylation ordephosphorylation reaction can be measured by a usual method well knownto those skilled in the art or by using a commercially available kit.Changes in cell growth activity can be used as the detection marker fora cell culture system. Changes in cell growth activity can be measuredby the MTT method or a method using tritium-labeled thymidine.Quantitative and/or qualitative changes in cell surface antigens can bedetected by detecting the bound amount of a fluorescence-labeledspecific antibody, using a flow cytometer or fluorescence microscope.Furthermore, depending on downstream signaling, phosphorylation ofsubstrate proteins, and changes in the concentration of secondmessengers such as cAMP and Ca²⁺ can also be measured as detectionmarkers. These methods are already well known, and the detection canalso be conducted by using generally used methods and kits. Reportergene assays using activity of, for example, luciferase, chloramphenicolacetyltransferase, and β-galactosidase can also be utilized for thedetection of downstream gene expression regulated by the chimericreceptors. As described above, screening by simultaneous and/orcontinuous measurement of these multiple distinct markers is one of theefficient and simple screening methods provided by the presentinvention. Due to the ease in detection, cell-based detection markersare preferable, and especially preferable is a method that uses cellgrowth activity as a detection marker.

Furthermore, in the methods of the present invention, thesignal-transducing domain derived from one of the two or more receptorsof interest can be used as it is. The other receptor can be chimericcomprising the above-mentioned signal-transducing domain and a differentextracellular domain. Chimeric receptors may have partial sequences oftwo or more distinct receptors. Alternatively, they may comprise thewhole receptor of one of two or more distinct receptors, plus a wholecompletely different receptor, or a portion thereof. For example,chimeric receptors can have the extracellular domain of a cell membranereceptor and the intracellular domain of another cell membrane receptor,and can also have the ligand-binding domain of a nuclear receptor andthe DNA-binding domain of another nuclear receptor. By using multiplechimeric receptors having a common DNA-binding domain and a differentligand-binding domain, the effect on the multiple receptors can besimultaneously detected. Furthermore, chimeric receptors having thewhole molecule or intracellular domain of a cell membrane receptor andthe whole molecule or ligand-binding domain of an intracellular receptorcan also be used. In this case, the intracellular domain of thischimeric receptor derives from a cell membrane receptor and theextracellular domain derives from an intracellular receptor. Therefore,a ligand inducing multimerization, preferably dimerization, by bindingto the intracellular receptor, can be detected by using a detectionmarker induced by the multimerization or dimerization of the cellmembrane receptor used.

The extracellular domain used for the chimeric receptor of a cellmembrane receptor, may be any domain other than the signal-transducingdomain of the receptor, and may be the whole extracellular domain, or aportion thereof. The whole extracellular domain may preferably be usedas it may properly reflect the biological activity. In the case of usingpartial domains of a receptor's extracellular domain, the ligand-bindingdomain, or a membrane-proximal domain having 20 or more amino acidresidues, preferably 50 or more residues, or more preferably 100 or moreresidues can be used for the construction of the chimeric receptor. Asthe extracellular domain utilized for the construction of a chimericreceptor, any partial sequence can be used, as long as themultimerization, preferably, dimerization, of the signal-transducingdomain is induced, and changes in the detection marker are also induced.In this case, there may a substitution, deletion, insertion, or additionto the amino acids constituting the extracellular domain. One methodwell known to those skilled in the art for preparing functionallyequivalent proteins is to introduce mutations into proteins. Forexample, those skilled in the art can construct such a protein by usingsite-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152,271-275; Zoller, M J, and Smith, M.(1983) Methods Enzymol. 100, 468-500;Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456; Kramer W, andFritz H J (1987) Methods Enzymol. 154, 350-367; Kunkel, T A (1985) ProcNatl Acad Sci USA. 82, 488-492; Kunkel (1988) Methods Enzymol. 85,2763-2766), etc.

It is thought that the number of amino acid residues mutated in such amutant protein is usually 50 or less, preferably 30 or less, morepreferably 20 or less, even more preferably 10 or less, further morepreferably 5 or less, or even further more preferably 3 or less.

It is preferable to mutate an amino acid residue into one that allowsthe properties of the amino acid side-chain to be conserved. Examples ofproperties of amino acid side chains include: hydrophobic amino acids(A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q,G, H, K, S, T), and amino acids comprising the following side chains:aliphatic side-chains (G, A, V, L, I, P); hydroxyl group-containingside-chains (S, T, Y); sulfur atom-containing side-chains (C, M);carboxylic acid- and amide-containing side-chains (D, N, E, Q);base-containing side-chain (R, K, H); and aromatic-containingside-chains (H, F, Y, W) (The letters within parentheses indicate theone-letter codes of amino acids).

It is well known that a protein having deletion, addition, and/orsubstitution of one or more amino acid residues in the sequence of theprotein can retain the original biological activity (Mark, D. F. et al.Proc. Natl. Acad. Sci. U.S.A. 81:5662-5666 (1984); Zoller, M. J. andSmith, M. Nucleic Acids Res. 10:6487-6500 (1982); Wang, A. et al.Science 224:1431-1433; Dalbadie-McFarland, G. et al. Proc. Natl. Acad.Sci. U.S.A. 79:6409-6413 (1982)).

The transmembrane domain utilized for the construction of a chimericreceptor can derive from the receptor from which the extracellular orintracellular domain derived. Furthermore, the transmembrane domain canderive from the both these receptor molecules utilized for theconstruction of the chimeric receptor, or can also derive from cellmembrane receptors other than those utilized for the construction. Sincemultiple chimeric receptors can easily be made, the transmembrane domainof chimeric receptors may preferably derive from receptors used as theintracellular domain for the construction of the chimeric receptor.

The intracellular domains of chimeric receptors used in the presentinvention are not restricted, as long as ligand-induced changes inphenotypes can be detected. For example, the intracellular domain ofG-CSF, EPO, EGF, or TPO receptor can be used, utilizing as a marker thecell growth activity induced by stimulation of these receptors. Forexample, a chimeric receptor having the extracellular domain of thegrowth hormone receptor and the intracellular domain of the G-CSFreceptor, has been shown to induce cell growth depending on growthhormone stimulation (Fuh, G. Science (1992) 256, 1677-1680).

It is preferable to use the signaling domain of mouse G-CSF receptor forthe construction of chimeric receptors, as the structure and function ofthe receptor have been studied in detail. The mouse G-CSF receptorconsists of 813 amino acid residues, and a single transmembrane domainseparates the extracellular and intracellular domains (Fukunaga, R. Cell(1990) 61, 341-350). It has also been shown that when the G-CSF receptoris expressed in FDC-P1, a myelocyte precursor cell line, and Ba/F3, apro-B cell line, the expressed receptor can transmit agrowth-stimulating signal, and a G-CSF-dependent proliferative activityis seen. Furthermore, it has also been shown that the intracellular 76amino acid residues of the G-CSF receptor are essential for thetransmission of the G-CSF growth stimulation (Fukunaga, R. EMBO J.(1991) 10, 2855-2865). By constructing a chimeric receptor containingthe 76 amino acid residues as the signal-transducing domain andexpressing the chimeric receptor in Ba/F3 cells, it is possible to usecell growth activity as a detection marker.

It has been shown that the deletion of downstream amino acid residuesafter the 716th residue of human G-CSF receptor restricts theinternalization of the receptor, resulting in enhanced signaltransduction efficiency following G-CSF stimulation due to the increasednumber of receptors on the cell surface (Melissa G., Blood (1999) 93,440-446). This deleted domain contains a motif called Box3, which ispresumed to be crucial for the receptor's internalization. Box1 and box2essential for the signal transduction are however retained. Therefore,it can be presumed that even in the mouse G-CSF receptor, it may bepossible to enhance the signal transduction efficiency following G-CSFstimulation by deleting the domain containing box3, but not box2.

Any cell line can be used to express a chimeric receptor, if the cellline can respond to the ligand only when the chimeric receptor isexpressed, and if the ligand does not cause phenotypic changes to thecell lines in which the chimeric receptor is not expressed. When cellgrowth activity is utilized as a detection marker, it is preferable touse cell lines that die in the absence of the ligand in order toincrease detection sensitivity. Particularly, cytokine-dependent celllines are useful, since they can easily be passaged. For example,IL-2-dependent CTLL-2 cells, IL-3-dependent 32D cells, FDC-P1 cells andBa/F3 cells can be used. These cell lines have the characteristic ofdieing in day-2 or -3 after removing cytokines, such as IL-2 or -3required for the growth, from the culture media. It is preferable to useFDC-P1 or Ba/F3 cells expressing a chimeric receptor comprising theintracellular domain of mouse G-CSF receptor. As hosts used in themethods of the present invention, not only animal cell lines but alsoyeast and Escherichia coli can be used. When ligand-mediated receptordimerization is used for the detection marker, for example, thetwo-hybrid system utilizing chimeric receptors can be used.Specifically, by constructing a gene the encodes a chimeric receptorcomprising the activation domain of yeast GAL4 and a receptor, or achimeric receptor comprising the ligand-binding domain of GAL4 and areceptor, and expressing in yeast, the expression of a reporter gene inthe yeast can be monitored in culture in the presence of test samples.The activities of many different substances on multiple cell membranereceptors can simultaneously be assayed by constructing multiplechimeric receptors from different receptors.

Furthermore, the cell lines of the present invention can be modified toimprove the screening sensitivity. The sensitivity of the cells can beincreased, for example, by expressing chimeric receptor molecules usingan appropriate regulatory domain and a polyadenylation signal so thatthe expression of chimeric receptor molecules will increase, or byreplacing the mRNA instability signals with stable ones. Chimericreceptor genes in which the domain flanking the initiation codon hasbeen modified into the Kozak's consensus sequence (CCACC) may also beused. Furthermore, it is possible to easily obtain cell lines with ahigh expression by combining with a suitable selection marker. Forexample, well known is a method in which dihydrofolate reductase (DHFR)is used as a marker for cell lines that lack the DHFR gene, andmethotrexate is used to inhibit DHFR to obtain a cell line that highlyexpresses an objective gene. Also known is a method that uses thethymidine kinase gene that lacks the promoter as a selection marker toselect a cell line that highly expresses a gene of interest.Furthermore, for example, it is also possible to specifically selectcells showing a high expression by using a cell sorter after binding aflorescence-labeled anti-receptor antibody or coexpressing with greenfluorescence protein (GFP), etc. Modification of the receptor'smetabolic process may also provide a highly sensitive detection system.For example, it is known that mouse G-CSF receptor lacking itsC-terminus internalizes less efficiently, resulting in the enhancedexpression. Since proteins having high contents of proline, glutamicacid, serine, and threonine generally are thought to degrade fast,mutating amino acid sequences to reduce such residues may also be usefulto enhance expression.

In the methods of the present invention, cell lines expressing severaldifferent chimeric receptors with different extracellular domains can beused if the intracellular domain of the receptors is the same, or if thechimeric receptors induce the same phenotypic change upon ligandbinding. The cell lines can induce changes in their phenotypes uponbinding to multiple different types of ligands. When the cell lines arecultivated in the presence of a test sample, changes in the cell'sphenotypes, for example, cell growth rate, can be detected. In thisdetection, phenotypic changes in the cultured cell indicate that thetest sample is a ligand for at least one of the receptors havingmultiple extracellular domains. The test samples may be mixed togetherand it is also possible to simultaneously detect a mixture of naturalsubstances.

The methods of the present invention can be performed by preparingchimeric receptors comprising a common intracellular domain for each ofthe objective two or more different types of receptors, and preparingcells that separately express each of the chimeric receptors, and mixingthese cells when performing the detection. The number of cells mixed isnot especially critical as long as the cell number is sufficient toappropriately observe the reactivity to a ligand. When the naturalligand is known, the suitability of the assay system can be evaluatedusing the natural ligand. The number of cells used is preferably 10 ormore per well, more preferably 100 or more per well, even morepreferably 1000 or more per well. Since a high concentration of cellsdecreases the detection sensitivity of cell growth activity, the cellconcentration used is preferably 1×10⁷/mL or less, and more preferably1×10⁶/mL or less. Although the assay of the cell growth rate can beusually carried out in 24-well or 96-well plates well known to thoseskilled in the art, the assay is not restricted by the number of wellsof plates used. It is also possible to use plates having 384 wells. Inthat case, using approximately ¼ of the number of cells used for a96-well plate is recommended. The detection is possible after one day ormore, but preferably 2 to 4 days, more preferably 3 days. The number ofcell line types is not critical as long as the phenotypic changes can bedetected upon ligand binding. However, the number of types is preferablytwo or more, more preferably three or more, even more preferably five ormore, and most preferably ten or more. The specificity of ligand samplescan be determined by using ligand-dependent cells or by detecting theirreactivity to individually cultured cells expressing a single chimericreceptor, based on the number of receptors of interest.

The receptor DNA of the present invention can be prepared using methodsknown in the art. For example, a cDNA library can be constructed fromcells expressing a protein of the present invention and hybridizationcan be conducted using a part of the DNA sequence of interest, which canbe found in literature, as a probe. The cDNA library may be prepared,for example, according to the method described by Sambrook J. et al.(Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)), orinstead, commercially available DNA libraries may be used.Alternatively, a DNA of the present invention can be obtained bypreparing RNA from cells expressing a protein of the present invention,synthesizing cDNA therefrom using a reverse transcriptase, synthesizingoligo-DNA based on a DNA sequence of interest, and amplifying the cDNAencoding a receptor by PCR using the oligo-DNA as primers.

A desired DNA fragment is prepared from the obtained PCR products andlinked to a vector DNA. The recombinant vector is used to transform E.coli and such, and the desired recombinant vector is prepared from aselected colony. Vector DNA well known to those skilled in the art(pUC19, pBluescript, etc.) can be used for harboring the DNA fragment.Escherichia coli strains well known to those skilled in the art (DH5α,JM109, etc.) can be used. The nucleotide sequence of the desired DNA canbe verified by conventional methods, such as dideoxynucleotide chaintermination (Sambrook, J. et al., Molecular Cloning, Cold Spring HarborLaboratory Press (1989)). The nucleotide sequence of the desired DNA canbe determined by using automated DNA sequencers such as DNA SequencerPRISM 377 and DNA Sequencer PRISM 310 (Perkin-Elmer), etc.

A DNA of the invention may be designed to have a sequence that isexpressed more efficiently by taking into account the frequency of codonusage in the host used for expression (Grantham, R. et al., NucleicAcids Res. 9:r43-74 (1981)). The DNA of the present invention may bealtered by a commercially available kit or a conventional method. Forinstance, the DNA may be altered by digestion with restriction enzymes,insertion of a synthetic oligonucleotide or an appropriate DNA fragment,addition of a linker, or insertion of the initiation codon (ATG) and/orthe stop codon (TAA, TGA, or TAG), etc.

To express chimeric receptor molecules, an expression vector, whichcomprises DNA encoding the chimeric receptor under the control ofregulatory sequences such as enhancers/promoters, is constructed. Then,the expression vector is used to simultaneously transform host cells andthe chimeric receptor is expressed in the cells. For receptors forming aheterodimer, simultaneous transformation with expression vectorsencoding each of the subunits can be done, or an expression vectorencoding multiple subunits can be prepared and used for thetransformation.

Useful promoters regularly used for expression in mammalian cells can beused. For example, human polypeptide chain elongation factor-1α (HEF-1α)is preferably used. pEFBOS (Mizushima, S. et al. (1990) Nuc. Acid Res.18, 5322) is an example of expression vectors comprising the HEF-1αpromoter. Furthermore, mammalian cell promoters and promoters ofviruses, including cytomegalovirus, retrovirus, polyomavirus,adenovirus, simian virus 40 (SV40), and such can be used for the methodsof the present invention. For example, the SV40 promoter can be easilyutilized by the method of Mulligan et al. (Nature (1990) 277, 108).

To introduce genes into host cells, expression vectors can contain thefollowing selection marker genes; phosphotransferase APH(3′) II or I(neo) gene, thymidine kinase gene, Escherichia coli xanthine-guaninephosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (DHFR)gene, etc.

For introducing genes into hosts, methods well known to those skilled inthe art, for example, the calcium phosphate method (Chen, C. et al.(1987) Mol. Cell. Biol. 7, 2745-2752), lipofection (Felgner, PL. et al.(1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417), and electroporation(Potter, H. (1988) Anal. Biochem. 174, 361-373) can be used. For themethods of the present invention, the electroporation method using aGene Transfer Equipment (Gene Pulser, Bio-Rad) can be used.

In the methods of the present invention, a further step, whereinbiological activity is detected by contacting a test sample with one ofthe two or more receptors used for the screening, may be included todetect the specificity of a test sample towards a receptor.

Furthermore, the present invention provides kits for the abovescreening. As components, the kits of the present invention contain (a)two or more kinds of receptors comprising (i) a commonsignal-transducing domain, and (ii) a domain other than thesignal-transducing domain, wherein said domain derives from the samereceptor from which the common domain of (i) is derived and/or adifferent receptor; (b) DNA encoding two or more kinds of receptorscomprising (i) a common signal-transducing domain, and (ii) a domainother than the signal-transducing domain, wherein said domain derivesfrom the same receptor from which the common domain of (i) is derivedand/or a different receptor; (c) a cell expressing two or more kinds ofreceptors comprising (i) a common signal-transducing domain, and (ii) adomain other than the signal-transducing domain, wherein said domainderives from the same receptor from which the common domain of (i) isderived and/or a different receptor. By setting up the above screeningsystem using the kit of the present invention, the screening of a ligandcan be efficiently done.

It is anticipated that substances (containing ligands) isolated by thescreenings of the present invention can be used as pharmaceutical agentsfor the treatment and prevention of various diseases, depending on theirbiological activity. For example, it is expected that such a substancecan be used as a pharmaceutical agent for the treatment of anemia if itis a ligand for the EPO receptor, for the treatment of neutropenia if itis a ligand for the G-CSF receptor, for the treatment ofthrombocytopenia if it is a ligand for the TPO receptor, for thetreatment of diabetes if it is a ligand for the insulin receptor, forimmunostimulation if it is a ligand for the Flt-3 ligand receptor, forstimulation of wound healing if it is a ligand for the PDGF receptor,for the treatment of viral diseases if it is a ligand for the IFN-α/βreceptors, for the treatment of obesity if it is a ligand for the leptinreceptor, for the treatment of short statue if it is a ligand for thegrowth hormone (GH) receptor, for immunosuppression (for example, forthe treatment of inflammatory bowel disease and rheumatoid arthritis) ifit is a ligand for the interleukin (IL)-10 receptor, for the treatmentof short statue if it is a ligand for the insulin-like growth factor(IGF)-I receptor, for the treatment of leukemia if it is a ligand forthe leukemia inhibitory factor (LIF) receptor, and for the treatment ofobesity (or of amyotrophic lateral sclerosis) if it is a ligand for theciliary neurotrophic factor (CNTF) receptor.

Such a substance or ligand can be as a pharmaceutical agent for humansand other mammals, such as mice, rats, guinea-pigs, rabbits, chicken,cats, dogs, sheep, pigs, cattle, monkeys, baboons, and chimpanzees.Specifically, it can either itself be directly administered to subjectsor it can be formulated using known pharmaceutical preparation methodsfor administration. For example, according to the need, the substancesor ligands can be taken orally, as sugar coated tablets, capsules,elixirs, and microcapsules; or non-orally, in the form of injections ofsterile solutions or suspensions with water or any otherpharmaceutically acceptable liquid. For example, the substances orligands can be formulated by mixing appropriately with pharmacologicallyacceptable carriers or medium, such as, sterilized water, physiologicalsaline, plant-oil, emulsifiers, suspending agents, surfactants,stabilizers, flavoring agents, excipients, vehicles, preservatives, andbinders, in a unit dose form required for generally accepted drugimplementation. The amount of active ingredient in these preparationsmakes a suitable dosage within the indicated range acquirable.

Examples of additives that can be mixed for tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum, and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin, and alginic acid; lubricants such as magnesiumstearate; sweeteners such as sucrose, lactose, or saccharin; flavoringagents such as peppermint, Gaultheria adenothrix oil, and cherry. Whenthe unit dosage form is a capsule, a liquid carrier, such as oil, canalso be included in the above ingredients. Sterile composites forinjections can be formulated following normal drug implementations usingvehicles such as distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannnose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (TM)and HCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizer or may be formulated with a buffer such as phosphate bufferand sodium acetate buffer, a pain-killer such as procaine hydrochloride,a stabilizer such as benzyl alcohol, phenol, or an anti-oxidant. Theprepared injection is generally filled into a suitable ampule.

Methods well known to one skilled in the art may be used to administer apharmaceutical compound to patients, for example as intraarterial,intravenous, subcutaneous injections and also as intranasal,transbronchial, intramuscular, percutaneous, or oral administrations.The dosage varies according to the body-weight and age of a patient, andthe administration method; however, one skilled in the art can suitablyselect the dosage.

Although varying according to the symptoms and such, the dose may begenerally in the range of about 0.1 mg to about 500 mg, preferably about1.0 mg to about 100 mg, and more preferably about 1.0 mg to about 20 mgper day for adults (body weight: 60 kg) in the case of an oraladministration.

Although varying according to the subject, target organ, symptoms, andmethod of administration, a single dose of a compound for parenteraladministration is advantageous, for example, when administeredintravenously to normal adults (60 kg body weight) in the form ofinjection, in the range of about 0.01 mg to about 30 mg, preferablyabout 0.1 mg to about 20 mg, and more preferably about 0.1 mg to about10 mg per day. Doses converted to 60 kg body weight or per body surfacearea can be administered to other animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequences of the used chimeric receptors.The extracellular domains were derived from various human receptors, andthe intracellular domains from mouse G-CSF receptor (from the 602nd tothe 812th amino acid residues of mouse G-CSF receptor).

FIG. 2 depicts the response of the chimeric EPO receptor-expressingFDC-P1 cell line F#14 against human EPO. An evident growth reactionagainst human EPO was observed at concentrations of 10 pg/mL or more.

FIG. 3 depicts the response of the chimeric EPO receptor-expressingBa/F3 cell line EPG against human EPO. An evident growth reactionagainst human EPO was observed at concentrations of 10 pg/mL or more.

FIG. 4 depicts the response of the chimeric TPO receptor-expressingBa/F3 cell line TPG against human TPO. An evident growth reactionagainst human TPO was observed at concentrations of 100 pg/mL or more.

FIG. 5 depicts the response of the chimeric G-CSF receptor-expressingBa/F3 cell line GFG against human G-CSF. An evident growth reactionagainst human G-CSF was observed at concentrations of 10 pg/mL or more.

FIG. 6 depicts the response of the chimeric Flt-3 receptor-expressingBa/F3 cell line FLG against human Flt-3 ligand. An evident growthreaction against human Flt-3 ligand was observed at concentrations of300 pg/mL or more.

FIG. 7 depicts the response of the chimeric insulin receptor-expressingcell line ING against human insulin.

FIG. 8 depicts the response of the chimeric PDGF receptor-expressingBa/F3 cell line PDG against human PDGF-BB. An evident growth reactionagainst human PDGF-BB was observed at concentrations of 30 ng/mL ormore.

FIG. 9 depicts the response of the chimeric IFNα receptor-expressingBa/F3 cell line IFG against human IFNα. An evident growth reactionagainst human IFNα was observed at concentrations of 200 U/mL or more.

FIG. 10 depicts the amino acid sequences of the used chimeric receptormolecules. The extracellular domains were derived from various humanreceptors, and the intracellular domains from mouse G-CSF receptor (fromthe 602nd to the 812th amino acid residues of mouse G-CSF receptor).

FIG. 11 depicts the response of the chimeric leptin receptor-expressingBa/F3 cell line LPG against human leptin. An evident growth reactionagainst human leptin was observed at concentrations of 0.2 ng/mL ormore.

FIG. 12 depicts the response of the chimeric GH receptor-expressingBa/F3 cell line GHG against human GH. An evident growth reaction againsthuman GH was observed at concentrations of 0.3 μIU/mL or more.

FIG. 13 depicts the response of the chimeric IL-10 receptor-expressingBa/F3 cell line 10G against human IL-10. An evident growth reactionagainst human IL-10 was observed at concentrations of 0.5 ng/mL or more.

FIG. 14 depicts the response of the chimeric IGF-I receptor-expressingBa/F3 cell line IGG against human IGF-I. An evident growth reactionagainst human IGF-I was observed at concentrations of 10 ng/mL or more.

FIG. 15 depicts the response of the chimeric LIF receptor-expressingBa/F3 cell line LIG against human LIF. An evident growth reactionagainst human LIF was observed at concentrations of 0.4 ng/mL or more,but not in the presence of human CNTF at concentrations of 10 ng/mL orless.

FIG. 16 depicts the response of the chimeric CNTF receptor-expressingBa/F3 cell line CNG against human CNTF. An evident growth reactionagainst human CNTF was observed at concentrations of 0.3 ng/mL or more.

FIG. 17 depicts the response of the chimeric PDGF receptor-expressingBa/F3 cell line PDG against human PDGF-BB.

An evident growth reaction against human PDGF-BB was observed atconcentrations of 0.5 ng/mL or more.

FIG. 18 depicts responses of cells to human EPO in individual and mixedcell culture systems. Open symbols indicate the responses of theindividually cultured established cell lines (4×10³ cells/well), andclosed circles those of the mixed culture of the four cell lines (eachcell line at 4×10³ cells/well, 16×10³ cells/well in total). Incomparison to the individual cultures (open upward triangle), a slightenhancement in the growth activity was observed for the mixed culture(closed circle) in the absence of human EPO. However, the response tohuman EPO was observed to be similar between those at the concentration1 to 10 pg/mL, where the response was observed to start, and at maximumstimulation.

FIG. 19 depicts the responses of cells to human TPO in individual andmixed culture systems. Open symbols indicate the responses of theindividually cultured established cell lines (4×10³ cells/well), andclosed circles those of the mixed culture of the four cell lines (eachcell line at 4×10³ cells/well, 16×10³ cells/well in total). Incomparison to the individual cultures (open diamond), a slightenhancement in the growth activity was observed for the mixed culture(closed circle) in the absence of human TPO. However, the response tohuman TPO was observed to be similar between those at the concentration100 to 300 pg/mL, where the response was observed to start, and atmaximum stimulation.

FIG. 20 depicts the responses of cells to human G-CSF in individual andmixed culture systems. Open symbols indicate the responses of theindividually cultured established cell lines (4×10³ cells/well), andclosed circles those of the mixed culture of the four cell lines (eachcell line at 4×10³ cells/well, 16×10³ cells/well in total). Incomparison to the individual culture (open downward triangle), a slightenhancement in the growth activity was observed for the mixed culture(closed circle) in the absence of human G-CSF. However, the response tohuman G-CSF was observed to be similar between those at theconcentration 2 to 5 pg/mL, where the response was observed to start,and at maximum stimulation.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below by way ofExamples, but should not be construed as being limited to theseExamples.

EXAMPLE 1 Establishment of Cell Lines Expressing Chimeric Receptors

(1-1) Construction of Mammalian Cell Expression Plasmid Vectors, PCOS-G,pCOS2, and pCV

Mammalian cell plasmid expression vector pCOS-G was constructed byreplacing the polyadenylation signal of pCOS1 (see InternationalPublication No. WO98/13388, “Antibody against human parathormone relatedpeptides”) with that of the human G-CSF gene. The polyadenylation signalof human G-CSF gene was obtained by digesting pEF-BOS (Mizushima S. etal. (1990) Nuc. Acid Res. 18, 5322) with Xho I and Pvu II. Thepolyadenylation signal fragment was replaced into the Xho I/Aor51H Ifragment of pCOS1, and the resulting construct was dubbed PCOS-G.

Mammalian cell plasmid expression vector pCOS2 was constructed byreplacing the BamHI/Aor51H I fragment of pCOS1 with the BamHI/Aor51HIfragment of pEGFP-N1 (CLONTECH).

Mammalian cell plasmid expression vector pCV was constructed byreplacing the polyadenylation signal of pCOS1 with that of the humanG-CSF gene. The polyadenylation signal of human G-CSF gene was obtainedby digesting pEF-BOS with EcoRI and Xba I. The 3′ end of the fragmentwas blunted, and a BamHI site was attached to the 5′ end of thefragment. The resulting fragment was replaced as the poly (A) additionsignal into the BamHI/Aor51H I site of pCOS1, and was used as pCV.

(1-2) Erythropoietin (Hereinafter, Referred to as EPO) Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the extracellular domain (from the 1st to the 249th amino acidresidues; Jones, SS. et al. (1990) Blood, 76, 31-35) of human EPOreceptor, and the transmembrane and intracellular domains (from the602nd to the 813th amino acid residues; Fukunaga, R. et al. (1990) Cell61, 341-350) of mouse G-CSF receptor. The cDNA was inserted downstreamof the EF1α promoter of the mammalian expression vector pCOS-G toconstruct chimeric receptor-expression vector EG/pCOS-G. EG/pCOS-G waslinearized using Pvu I (Takara Shuzo), extracted with phenol andchloroform, and purified by ethanol precipitation.

The linearized expression vector was introduced into mouse FDC-P1 cells(ATCC No. CRL-12103) using an electroporation apparatus (Gene Pulser:Bio Rad). The FDC-P1 cells were washed twice with Dulbecco's PBS(hereinafter referred to as PBS), and were suspended in PBS to give acell density of about 1×10⁷ cells/mL. 10 μg of the linearized expressionvector DNA was added to 0.8 mL of the suspension, transferred into acuvette for electroporation (Bio Rad), and pulsed with a capacitance of250 μF at 0.35 kV.

After standing still at room temperature for about 10 min, the cellstreated by electroporation were suspended in Media A that wassupplemented with 1 ng/mL human EPO (prepared from genetic recombinantCHO cells), and then were seeded on a 96-well microtiter plate (flatbottom, Falcon) at 100 μL/well. After culturing in a CO₂ incubator (CO₂concentration, 5%) for about six hours, 100 μL/well of Media Asupplemented with 1 ng/mL EPO and 1 mg/mL GENETICIN (GIBCO) was added tothe plate, and was cultured in a CO₂ incubator (CO₂ concentration, 5%).RPMI1640 (GIBCO) supplemented with 10% fetal bovine serum (GIBCO), 100U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO) was used as Media A.About one week after the start of the culture, cells were observed undera microscope, and cells were collected from wells with a single colony.The collected cells were subcultured in Media A containing 1 ng/mL humanEPO. The titer of human EPO used in this Example was 270,000 IU/mg.

The cells were washed twice with Media A, and then suspended in Media Ato give a cell density of 2×10⁵ cells/mL. 50 μl/well of the cellsuspension and 50 μl/well of human EPO, appropriately diluted with MediaA, were dispensed into wells of a 96-well microtiter plate (flat bottom,Falcon), and were cultured for 24 hours in a CO₂ incubator (CO₂concentration, 5%). After the culture, 10 μl/well WST-8 reagent (CellCounting Kit-8; DOJINDO LABORATORIES) was added, and the plate wasincubated for five hours in a CO₂ incubator (CO₂ concentration, 5%).Then, absorbance at a measurement wavelength of 450 nm and a controlwavelength of 655 nm was measured using microplate reader (Model 3550,Bio Rad). Based on the cell growth activity determined by the number ofviable cells as the index by plotting the absorbance measured after thefive-hour incubation on the vertical axis and the concentrations ofhuman EPO on the horizontal axis, cell line F#14 that had a highsensitivity to human EPO was selected (FIG. 2).

The linearized expression gene vector was introduced into mouse Ba/F3cells (purchased from RIKEN; Cell No.: RCB0805) using an electroporationapparatus (Gene Pulser, BIO Rad). The Ba/F3 cells were washed twice withDulbecco's PBS (hereinafter referred to as PBS), and then were suspendedin PBS to give a cell density of about 1×10⁷ cells/mL. 10 μg of thelinearized expression vector DNA was added to 0.8 mL of this suspension,transferred into a cuvette (Bio Rad) for electroporation, and pulsedwith a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in Media A containing 1 ng/mLhuman EPO, and then were seeded on a 96-well microtiter plate (flatbottom, Falcon) at 100 μl/well. After culturing the cells for about fivehours in a CO₂ incubator (CO₂ concentration, 5%), 100 μL/well Media Athat contained 1 ng/mL human EPO and 1 mg/mL GENETICIN (GIBCO) wasadded, and the cells were cultured in a CO₂ incubator (CO₂concentration, 5%). RPMI1640 (GIBCO) supplemented with 10% fetal bovineserum (GIBCO), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO)was used as Media A. About one week after the start of the cultivation,cells were observed under a microscope, cells were collected from wellswith a single colony, and were subcultured in Media A containing 1 ng/mLhuman EPO. The cells were washed twice with Media A, and were suspendedin Media A to give a cell density of 2×10⁵ cells/mL. 50 μl/well of thecell suspension and 50 μl/well of human EPO appropriately diluted withMedia A were dispensed into the wells of a 96-well microtiter plate(flat bottom, Falcon), and were cultured for 24 hours in a CO₂ incubator(CO₂ concentration, 5%). After the culture, 10 μl/well WST-8 reagent(Cell Counting Kit-8; DOJINDO LABORATORIES) was added, and the plate wasincubated for five hours in a CO₂ incubator (CO₂ concentration, 5%).After the incubation, absorbance at a measurement wavelength of 450 nmand a control wavelength of 655 nm was measured using microplate reader(Model 3550, Bio Rad). Based on the cell growth activity determined bythe number of viable cells as an index by plotting the absorbancemeasured after the five-hour incubation on the vertical axis and theconcentrations of human EPO on the horizontal axis, cell line B#20 thathad a high sensitivity to human EPO was selected, and was used aschimeric EPO receptor-expressing cell line EPG (FIG. 3). Similar growthresponses to human EPO were observed by using FDC-P1 and Ba/F3 as hostcells.

(1-3) Thrombopoietin (Hereinafter Referred to as TPO) Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the extracellular domain (from the 1st to the 491st amino acidresidues; Vigon, I. et al. (1992) Proc. Natl. Acad. Sci. USA 89,5640-5644) of human TPO receptor, and the transmembrane andintracellular domains (from the 602nd to the 813th amino acid residues;Fukunaga, R. et al. (1990) Cell 61, 341-350) of mouse G-CSF receptor.The cDNA was inserted downstream of the EF1α promoter of the mammalianexpression vector pCOS-G to construct chimeric receptor-expressionvector TG/pCOS-G. TG/pCOS-G was linearized with Pvu I (Takara Shuzo),extracted with phenol and chloroform, and purified by ethanolprecipitation.

The linearized expression gene vector was introduced into mouse Ba/F3cells (purchased from RIKEN; Cell No.: RCB0805) using an electroporationapparatus (Gene Pulser: Bio Rad) The Ba/F3 cells were washed twice withDulbecco's PBS (hereinafter referred to as PBS), and then were suspendedin PBS to give a cell density of about 1×10⁷ cells/mL. 10 μg of thelinearized expression vector DNA was added to 0.8 mL of this suspension,transferred into a cuvette (Bio Rad) for electroporation, and was pulsedwith a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in Media A, and were seeded ona 96-well microtiter plate (flat bottom, Falcon) at 100 μL/well. 100μL/well Media A that contained 2 ng/mL human TPO (R&D Systems) and 1mg/mL GENETICIN (GIBCO) was added, and the cells were cultured in a CO₂incubator (CO₂ concentration, 5%). RPMI1640 (GIBCO) supplemented with10% fetal bovine serum (GIBCO), 100 U/mL penicillin, and 0.1 mg/mLstreptomycin (GIBCO) was used as Media A. About one week after the startof culture, the cells were observed under a microscope, cells werecollected from wells with a single colony and were subcultured in MediaA containing 1 ng/mL human TPO.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well cell suspension and50 μl/well of human TPO (R&D Systems) appropriately diluted with Media Awere dispensed into the wells of a 96-well microtiter plate (flatbottom, Falcon), and were cultured for 72 hours in a CO₂ incubator (CO₂concentration, 5%). After the culture, 10 μl/well WST-8 reagent (CellCount Reagent SF; Nacalai Tesque) was added, and the plate was incubatedfor two hours in a CO₂ incubator (CO₂ concentration, 5%). The absorbanceat a measurement wavelength of 450 nm and a control wavelength of 655 nmafter the 2 hours incubation was measured using microplate reader (Model3550, Bio Rad). Based on the cell growth activity determined by thenumber of viable cells as an index by plotting the absorbance measuredafter the two-hour incubation on the vertical axis and theconcentrations of human TPO on the horizontal axis, cell line TPG#219that had a high sensitivity to human TPO was selected and was used aschimeric TPO receptor-expressing cell line TPG (FIG. 4).

(1-4) Granulocyte-Colony Stimulating Factor (Hereinafter Referred to asG-CSF) Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the extracellular domain (from the −23rd to the 604th aminoacid residues; Fukunaga, R. et al. (1990) Proc. Natl. Acad. Sci. USA 87,8702-8706) of human G-CSF receptor, and the transmembrane andintracellular domains (from the 602nd to the 813th amino acid residues;Fukunaga, R. et al. (1990) Cell 61, 341-350) of mouse G-CSF receptor.The cDNA was inserted downstream of the HEF1α promoter of the mammalianexpression vector pCOS-G to construct chimeric receptor-expressionvector GG/pCOS-G. GG/pCOS-G was linearized with Pvu I (Takara Shuzo),extracted with phenol and chloroform, and purified by ethanolprecipitation.

The linearized expression gene vector was introduced into mouse Ba/F3cells using an electroporation apparatus (Gene Pulser: Bio Rad). TheBa/F3 cells were washed twice with Dulbecco's PBS (hereinafter referredto as PBS), and then were suspended in PBS to give a cell density ofabout 1×10⁷ cells/mL. 10 μg of linearized expression vector DNA wasadded to 0.8 mL of this suspension, transferred into a cuvette (Bio Rad)for electroporation, and was pulsed with a capacitance of 960 μF at 0.33kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in Media A, and were seeded ona 96-well microtiter plate (flat bottom, Falcon) at 100 μL/well. 100μL/well Media A containing 2 ng/mL human G-CSF was added, and the cellswere cultured in a CO₂ incubator (CO₂ concentration, 5%). RPMI1640(GIBCO) supplemented with 10% fetal bovine serum (Hyclone) 100 U/mLpenicillin, and 0.1 mg/mL streptomycin (GIBCO) was used as Media A.After about one week from the start of the culture, the cells wereobserved under a microscope, cells were collected from wells with asingle colony and were subcultured in Media A containing 10 ng/mL humanG-CSF. The human G-CSF used in this Example was prepared fromrecombinant CHO cells, and the titer of human G-CSF was 1.2×10⁸ IU/mg.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well cell suspension and50 μl/well of human G-CSF (prepared from recombinant CHO cells)appropriately diluted in Media A were dispensed into wells of a 96-wellmicrotiter plate (flat bottom, Falcon), and were cultured for 70 hoursin a CO₂ incubator (CO₂ concentration, 5%). After the culture, 10μl/well WST-8 reagent (Cell Count Reagent SF; Nacalai Tesque) was added,and pre-reaction absorbance at a measurement wavelength of 450 nm and acontrol wavelength of 655 nm was measured using microplate reader (Model3550, Bio Rad). The plate was incubated for two hours in a CO₂ incubator(CO₂ concentration, 5%), and post-reaction absorbance was measured asabove. Based on the cell growth activity determined by the number ofviable cells by plotting the amount of changes in absorbance after thetwo-hour incubation on the vertical axis and the concentrations of humanG-CSF on the horizontal axis, cell line GFG#342 that had a highsensitivity to human G-CSF was selected and used as chimeric G-CSFreceptor-expressing cell line GFG (FIG. 5).

(1-5) Flt-3/Flk-2 Ligand (Hereinafter Referred to as Flt-3 Ligand)Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the extracellular domain (from the 1st to the 541st amino acidresidues; Small, D. et al. (1994) Proc. Natl. Acad. Sci. USA. 91,459-463) of human Flt-3, and the transmembrane and intracellular domains(from the 602nd to the 813th amino acid residues; Fukunaga, R. et al.(1990) Cell 61, 341-350) of mouse G-CSF receptor. The cDNA was inserteddownstream of the EF1α promoter of the mammalian expression vector pCOS2to construct chimeric receptor-expression vector FLG/pCOS2. TheFLG/pCOS2 was linearized with Hpa I (Takara Shuzo), extracted withphenol and chloroform, and purified by ethanol precipitation.

The linearized expression gene vector was introduced into mouse Ba/F3cells using an electroporation apparatus (Gene Pulser: Bio Rad). TheBa/F3⁻ cells were washed twice with Dulbecco's PBS (hereinafter referredto as PBS), and then were suspended in PBS to give a cell density ofabout 1×10⁷ cells/mL. 10 μg of the linearized expression vector DNA wasadded to 0.8 mL of this suspension, transferred into a cuvette (Bio Rad)for electroporation, and pulsed with a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in Media A, and were seeded ona 96-well microtiter plate (flat bottom, Falcon) at 100 μL/well. 100μL/well Media A that contained 10 ng/mL human Flt-3 ligand (Genzyme) wasadded, and the cells were cultured in a CO₂ incubator (CO₂concentration, 5%). RPMI1640 (GIBCO) supplemented with 10% fetal bovineserum (Hyclone), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO)was used as Media A. About one week after the start of the culture, thecells were observed under a microscope, cells were collected from wellswith a single colony and subcultured in Media A containing 5 ng/mL humanFlt-3 ligand.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well cell suspension and50 μl/well of human Flt-3 ligand appropriately diluted in Media A weredispensed into wells of a 96-well microtiter plate (flat bottom,Falcon), and were cultured for 74 hours in a CO₂ incubator (CO₂concentration, 5%). After the culture, 10 μl/well WST-8 reagent (CellCount Reagent SF; Nacalai Tesque) was added, and pre-reaction absorbanceat a measurement wavelength of 450 nm and a control wavelength of 655 nmwas measured using microplate reader (Model 3550, Bio Rad). The platewas incubated for two hours in a CO₂ incubator (CO₂ concentration, 5%;humidity, 99.9%), and post-reaction absorbance was measured as above.Based on the cell growth activity determined by the number of viablecells by plotting the amount of change in absorbance after the two-hourincubation on the vertical axis and the concentrations of human Flt-3ligand on the horizontal axis, cell line FLG#102 that had a highsensitivity to human Flt-3 ligand was selected and used as chimericFlt-3 ligand receptor-expressing cell line FLG (FIG. 6).

(1-6) Insulin Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the extracellular domain (from the −27th to the 917th aminoacid residues; Ullrich, A. et al. (1985) Nature 313, 756-761) of humaninsulin receptor, and the transmembrane and intracellular domains (fromthe 602nd to the 813th amino acid residues,; Fukunaga, R. et al. (1990)Cell 61, 341-350) of mouse G-CSF receptor. The cDNA was inserteddownstream of the HEF1α promoter of the mammalian expression vector pCVto construct chimeric receptor-expression vector ING/pCv. ING/pCV waslinearized with Pvu I (Takara Shuzo) extracted with phenol andchloroform, and purified by ethanol precipitation.

The linearized expression gene vector was introduced into mouse Ba/F3cells using an electroporation apparatus (Gene Pulser: Bio Rad). TheBa/F3 cells were washed twice with Dulbecco's PBS (hereinafter referredto as PBS), and then were suspended in PBS to give a cell density ofabout 1×10⁷ cells/mL. 10 μg of the linearized expression, vector DNA wasadded to 0.8 mL of this suspension, transferred into a cuvette (Bio Rad)for electroporation, and pulsed with a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in Media A, and were seeded ona 96-well microtiter plate (flat,bottom, Falcon) at 100 μL/well. 100μL/well Media A containing 10 μg/mL human insulin (SIGMA) was added, andthe cells were cultured in a CO₂ incubator (CO₂ concentration, 5%).RPMI1640 (GIBCO) supplemented with 10% fetal bovine serum (Hyclone), 100U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO) was used as Media A.About one week after the start of the culture, the cells were observedunder a microscope, cells were collected from wells with a single colonyand subcultured in Media A containing 10 μg/mL human insulin.

The cells were washed twice with media without human insulin(hereinafter referred to as Media B), and were suspended in Media B togive a cell density of 5×10⁴ cells/mL. CHO-S-SFM II medium (GIBCO)prepared without the addition of human insulin was used as Media B. 100μl/well of the cell suspension, 80 μL/well of Media B, and 20 μl/well ofhuman insulin appropriately diluted in 10 mM HCl solution that contained0.1% bovine serum albumin (SIGMA) were dispensed into the wells of a96-well microtiter plate (flat bottom, Falcon), and were cultured for 72hours in a CO₂ incubator (CO₂ concentration, 5%). After the culture, 20μl/well WST-8 reagent (Cell Count Reagent SF; Nacalai Tesque) was added,and pre-reaction absorbance at a measurement wavelength of 450 nm and acontrol wavelength of 655 nm was measured using microplate reader (Model3550, Bio Rad). The plate was incubated for two hours in a CO₂ incubator(CO₂ concentration, 5%), and post-reaction absorbance was measured asabove. Based on the cell growth activity determined by the number ofviable cells by plotting the amount of change in absorbance after thetwo-hour incubation on the vertical axis and the concentrations of humaninsulin on the horizontal axis, cell line ING#139 that had a highsensitivity to human insulin was selected and used as chimeric insulinreceptor-expressing cell line ING (FIG. 7).

(1-7) Platelet-Derived Growth Factor (Hereinafter Referred to as PDGF)Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the β chain (from the 1st to the 531st amino acid residues;Gronwald, RGK. et al. (1988) Proc. Natl. Acad. Sci. USA. 85, 3435-3439)of human PDGF receptor, and the transmembrane and intracellular domains(from the 602nd to the 813th amino acid residues; Fukunaga, R. et al.(1990) Cell 61, 341-350) of mouse G-CSF receptor. The cDNA was inserteddownstream of the HEF1α promoter of the mammalian expression vector pCVto construct chimeric receptor-expression vector pCV-cPDGFR. pCV-cPDGFRwas linearized with Pvu I (Takara Shuzo), extracted with phenol andchloroform, and purified by ethanol precipitation.

The linearized expression gene vector was introduced into mouse Ba/F3cells using an electroporation apparatus (Gene Pulser: Bio Rad). TheBa/F3 cells were washed twice with Dulbecco's PBS (hereinafter referredto as PBS), and then were suspended in PBS to give a cell density ofabout 1×10⁷ cells/mL. 20 μg of the linearized expression vector DNA wasadded to 0.8 mL of this suspension, transferred into a cuvette (Bio Rad)for electroporation, and pulsed with a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in Media A, and were seeded ona 96-well microtiter plate (flat bottom, Falcon) at 100 μL/well. 100μL/well Media A containing 40 ng/mL human PDGF-BB (Genzyme) was added,and the cells were cultured in a CO₂ incubator (CO₂ concentration, 5%)RPMI1640 (GIBCO) supplemented with 10% fetal bovine serum (Hyclone), 100U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO) was used as Media A.About one week after the start of the culture, the cells were observedunder a microscope, cells were collected from wells with a single colonyand subcultured in Media A that contained 20 ng/mL human PDGF-BB.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well of the cellsuspension and 50 μL/well human PDGF-BB appropriately diluted in Media Awere dispensed into the wells of a 96-well microtiter plate (flatbottom, Falcon), and were cultured for 70 hours in a CO₂ incubator (CO₂concentration, 5%). After the culture, 10 μL/well WST-8 reagent (CellCount Reagent SF; Nacalai Tesque) was added, and pre-reaction absorbanceat a measurement wavelength of 450 nm and a control wavelength of 655 nmwas measured using microplate reader (Model 3550, Bio Rad). The platewas incubated for two hours in a CO₂ incubator (CO₂ concentration, 5%),and post-reaction absorbance was measured as above. Based on the cellgrowth activity determined by the number of viable cells by plotting theamount of change in absorbance after the two-hour incubation on thevertical axis and the concentrations of human PDGF-BB on the horizontalaxis, cell line PDG#35 that had a high sensitivity to human PDGF-BB wasselected and used as chimeric PDGF receptor-expressing cell line PDG(FIG. 8).

(1-8) Interferon (Hereinafter Referred to as IFN) α/β Receptor

Chimeric receptor cDNAs were constructed by linking cDNA fragmentsencoding extracellular domain of the α chain (AR1) (from the 1st to the436th amino acid residues; Uze, G. et al. (1990) Cell 60, 225-234) andthe α/β chain (AR2;β) (from the 1st to the 243rd amino acid residues;Novick, D. et al. (1994) Cell 77, 391-400) of human IFN α/β receptorwith the transmembrane and intracellular domains (from the 602nd to the813th amino acid residues; Fukunaga, R. et al. (1990) Cell 61, 341-350)of mouse G-CSF receptor, respectively. These cDNAs were inserteddownstream of the HEF1α promoter of the mammalian expression vector pCVto construct chimeric receptor-expression vectors, IFGα/pCV andIFGβ/pCV, respectively. Both IFGα/pCv and IFGβ/pCV were linearized withPvu I (Takara Shuzo), respectively, extracted with phenol andchloroform, and purified by ethanol precipitation.

The linearized expression gene vectors were introduced into mouse Ba/F3cells using an electroporation apparatus (Gene Pulser: Bio Rad). TheBa/F3 cells were washed twice with Dulbecco's PBS (hereinafter referredto as PBS), and then were suspended in PBS to give a cell density ofabout 1×10⁷ cells/mL. 10 μg each of linearized IFGα/pCV and IFGβ/pCV wasadded to 0.8 mL of the suspension, transferred into a cuvette (Bio Rad)for electroporation, and pulsed with a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 mm at room temperature, the cellswere suspended in Media A, and were seeded on a 96-well microtiter plate(flat bottom, Falcon) at 100 μL/well. 100 μL/well Media A containing1,000 U/mL human IFNα (CALBIOCHEM) was added, and the cells werecultured in a CO2 incubator (CO2 concentration, 5%). RPMI1640 (GIBCO)supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin,and 0.1 mg/mL streptomycin (GIBCO) was used as Media A. About one weekafter the start of the culture, the cells were observed under amicroscope, cells were collected from wells with a single colony andsubcultured in Media A containing 1,000 U/mL IFNα.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well cell suspension and50 μL/well of human IFNa appropriately diluted in Media A were dispensedinto the wells of a 96-well microtiter plate (flat bottom, Falcon), andwere cultured for 72 hours in a CO₂ incubator (CO₂ concentration, 5%).After the culture, 10 μL/well WST-8 reagent (Cell Count Reagent SF;Nacalai Tesque) was added, and pre-reaction absorbance at a measurementwavelength of 450 nm and a control wavelength of 655 nm was measuredusing microplate reader (Model 3550, Bio Rad). The plate was incubatedfor two hours in a CO₂ incubator (CO₂ concentration, 5%), andpost-reaction absorbance was measured as above. Based on the cell growthactivity determined by the number of viable cells by plotting the amountof changes in absorbance measured after the two-hour incubation on thevertical axis and the concentrations of human IFNα on the horizontalaxis, cell line IFG#A01 that had a high sensitivity to human IFNα wasselected and used as chimeric IFNα receptor-expressing cell line IFG.

The IFNα/β receptor has been known to form a heterodimer, not ahomodimer, to transmit the signal. According to the present experiment,even if the intracellular domain of a heterodimer forming receptor wasreplaced with that of a homodimer forming receptor (represented by theG-CSF receptor), the chimeric receptor induced signal transduction andthe response of ligands could be detected as the cell growing activity(FIG. 9).

(1-9) Leptin Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the extracellular domain (from the 1st to the 839th amino acidresidues; Tartaglia, L A. et al. (1995) Cell, 83, 1263-1271) of humanleptin receptor, and the transmembrane and intracellular domains (fromthe 602nd to the 813th amino acid residues; Fukunaga, R. et al. (1990)Cell 61, 341-350) of mouse G-CSF receptor. This cDNA was inserteddownstream of the HEF1α promoter of the mammalian expression vector pCVto construct chimeric receptor expression vector pCV-cLepR. pCV-cLepRwas linearized with Pvu I (Takara Shuzo), extracted with phenol andchloroform, and purified by ethanol precipitation.

The linearized expression gene vector was introduced into mouse Ba/F3cells (purchased from RIKEN; Cell No.: RCB0805) using an electroporationapparatus (Gene Pulser: Bio Rad) The Ba/F3 cells were washed twice withDulbecco's PBS (hereinafter referred to as PBS), and then were suspendedin PBS to give a cell density of about 1×10⁷ cells/mL. 10 μg of thelinearized expression vector DNA was added to 0.8 mL of this suspension,transferred into a cuvette (Bio Rad) for electroporation, and pulsedwith a capacitance of 960 μF at 0.33 kV.

After leaving standing for 10 min at room temperature, the cells treatedby electroporation were suspended in 50 mL Media A, and were seeded onfive 96-well microtiter plates (flat bottom, Falcon) at 100 μL/well. 100μL/well Media A containing 10 ng/mL human leptin (Genzyme) was added,and the cells were cultured in a CO₂ incubator (CO₂ concentration, 5%).RPMI1640 (GIBCO) supplemented with 10% fetal bovine serum (Hyclone), 100U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO) was used as Media A.About one week after the start of the culture, the cells were observedunder a microscope, cells were collected from wells of a single colonyand subcultured in Media A containing 10 ng/mL human leptin.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well of the cellsuspension and 50 μL/well of human leptin appropriately diluted in MediaA were dispensed into the wells of a 96-well microtiter plate (flatbottom, Falcon), and were cultured for 72 hours in a CO₂ incubator (CO₂concentration, 5%). After the culture, 10 μL/well WST-8 reagent (CellCount Reagent SF; Nacalai Tesque) was added, and pre-reaction absorbanceat a measurement wavelength of 450 nm and a control wavelength of 655 nmwas measured using microplate reader (Model 3550, Bio Rad). The platewas incubated for two hours in a CO₂ incubator (CO₂ concentration, 5%),and post-reaction absorbance was measured as above. Based on the cellgrowth activity determined by the number of viable cells by plotting theamount of changes in the absorbance measured after the two-hourincubation on the vertical axis and the concentrations of human leptinon the horizontal axis, cell line LPG#51 that had a high sensitivity tohuman leptin was selected, and was used as chimeric leptinreceptor-expressing cell line LPG. The subculture of the cell line wasconducted in the presence of 1 ng/mL human leptin. An obvious growthreaction against leptin could be observed at a concentration of 0.2ng/mL or more of human leptin (FIG. 11).

(1-10) Growth Hormone (Hereinafter Referred to as GH) Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the extracellular domain (from the 18th to the 246th amino acidresidues; Leung, D W. et al. (1987) Nature 330, 537-543) of human GHreceptor, and the transmembrane and intracellular domains (from the602nd to the 813th amino acid residues; Fukunaga, R. et al. (1990) Cell61, 341-350) of mouse G-CSF receptor. This cDNA was inserted downstreamof the EF1α promoter of the mammalian expression vector pCV to constructchimeric receptor-expression vector GHG/pCV. GHG/pCV was linearized withPvu I (Takara Shuzo), extracted with phenol and chloroform, and purifiedby ethanol precipitation.

The linearized expression gene vector was introduced into mouse Ba/F3cells (purchased from RIKEN; Cell No.: RCB0805) using an electroporationapparatus (Gene Pulser: Bio Rad). The Ba/F3 cells were washed twice withDulbecco's PBS (hereinafter referred to as PBS), and then were suspendedin PBS to give a cell density of about 1×10⁷ cells/mL. 10 μg of thelinearized expression vector DNA was added to 0.8 mL of this suspension,transferred into a cuvette (Bio Rad) for electroporation, and pulsedwith a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in 50 mL Media A, and wereseeded on five 96-well microtiter plates (flat bottom, Falcon) at 100μL/well. 100 μL/well Media A that contained 200 μIU/mL human GH(Genotropin(R): Pharmacia & Upjohn) was added, and the cells werecultured in a CO₂ incubator (CO₂ concentration, 5%). RPMI1640 (GIBCO)supplemented with 10% fetal bovine serum (HyClone), 100 U/mL penicillin,and 0.1 mg/mL streptomycin (GIBCO) was used as Media A. About one weekafter the start of the culture, the cells were observed under amicroscope, cells were collected from wells of a single colony andsubcultured in Media A containing 500 μIU/mL human GH.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well cell suspension and50 μL/well human GH appropriately diluted in Media A were dispensed intothe wells of a 96-well microtiter plate (flat bottom, Falcon), and werecultured for 72 hours in a CO₂ incubator (CO₂ concentration, 5%). Afterthe incubation, 10 μL/well WST-8 reagent (Cell Count Reagent SF; NacalaiTesque) was added, and pre-reaction absorbance at a measurementwavelength of 450 nm and a control wavelength of 655 nm was measuredusing microplate reader (Model 3550, Bio Rad). The plate was incubatedfor two hours in a CO₂ incubator (CO₂ concentration, 5%), andpost-reaction absorbance was measured as above. Based on the cell growthactivity determined by the number of viable cells by plotting the amountof change in the absorbance measured after the two-hour incubation onthe vertical axis and the concentrations of human GH on the horizontalaxis, cell line GHG#11 that had a high sensitivity to human GH wasselected, and was used as chimeric GH receptor-expressing cell line GHG.This cell line was subcultured in the presence of 20 μIU/mL human GH. Ata concentration of 0.3 μIU/mL or more, an obvious growth reactionagainst human GH was observed (FIG. 12).

(1-11) Interleukin 10 (Hereinafter Referred to as IL-10) Receptor

Chimeric receptor cDNAs were constructed by linking cDNA fragmentsencoding the α chain (IL-10Rα) (from the 1st to the 235th amino acidresidue; Liu, Y. et al. (1994) J. Immunol. 152, 1821-1829) and thehIL-10R subunit (CRFB4; IL-10Rβ) extracellular domain (from the 1st tothe 220th amino acid residues; Lutfalla, G. et al. (1993) Genomics 16,366-373) of human IL-10 receptor with the transmembrane andintracellular domains (from the 602nd to the 813th amino acid residues;Fukunaga, R. et al. (1990) Cell 61, 341-350) of mouse G-CSF receptor,respectively. These constructs were inserted downstream of the HEF1αpromoter of the mammalian expression vector pCV to construct chimericreceptor-expression vectors, pCV -cIL10Rα and pCV-cIL10Rβ, respectively.pCV-cIL10Rα and pCV-cIL10Rβ were linearized with Pvu I (Takara Shuzo),respectively, extracted with phenol and chloroform, and purified byethanol precipitation.

The linearized expression gene vectors were introduced into mouse Ba/F3cells, respectively, using an electroporation apparatus (Gene Pulser:Bio Rad). The Ba/F3 cells were washed twice with Dulbecco's PBS(hereinafter referred to as PBS), and then were suspended in PBS to givea cell density of about 1×10⁷ cells/mL. 10 μg each of linearizedexpression vector DNA, pCv-cIL10Rα and pCV-cIL10Rβ, was added to 0.8 mLof this suspension, transferred into a cuvette (Bio Rad) forelectroporation, and pulsed with a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in 50 mL Media A, and wereseeded on five 96-well microtiter plates (flat bottom, Falcon) at 100μL/well. 100 μL/well Media A that contained 10 ng/mL of human IL-10(Genzyme) was added, and the cells were cultured in a CO₂ incubator (CO₂concentration, 5%). RPMI1640 (GIBCO) supplemented with 10% fetal bovineserum (Hyclone), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO)was used as Media A. About one week after the start of the culture, thecells were observed under a microscope, cells were collected from wellswith a single colony and subcultured in Media A containing 5 ng/mL humanIL-10.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well cell suspension and50 μL/well human IL-10 appropriately diluted in Media A were dispensedinto wells of a 96-well microtiter plate (flat bottom, Falcon), and werecultured for 72 hours in a CO₂ incubator (CO₂ concentration, 5%). Afterthe culture, 10 μL/well WST-8 reagent (Cell Count Reagent SF; NacalaiTesque) was added, and pre-reaction absorbance at a measurementwavelength of 450 nm and a control wavelength of 655 nm was measuredusing microplate reader (Model 3550, Bio Rad). The plate was incubatedfor two hours in a CO₂ incubator (CO₂ concentration, 5%), andpost-reaction absorbance was measured as above. Based on the cell growthactivity determined by the number of viable cells by plotting the amountof changes in absorbance measured after the two-hour incubation on thevertical axis and the concentrations of human IL-10 on the horizontalaxis, cell line 10G#10 that had a high sensitivity to human IL-10 wasselected and used as chimeric IL-10 receptor-expressing cell line 10G.This cell line was subcultured in the presence of 1 ng/mL human IL-10.At a concentration of 0.5 ng/mL or more, an obvious growth reactionagainst human IL-10 was observed (FIG. 13).

(1-12) Insulin-Like Growth Factor I (Hereinafter Referred to as IGF-I)Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the extracellular domain (from the 1st to the 931st amino acidresidues; Ullrich, A. et al. (1986) EMBO J., 5, 2503-2512) of humanIGF-I receptor, and the transmembrane and intracellular domains (fromthe 602nd to the 813th amino acid residues; Fukunaga, R. et al. (1990)Cell 61, 341-350) of mouse G-CSF receptor. This construct was inserteddownstream of the HEF1α promoter of the mammalian expression vector pCVto construct chimeric receptor-expression vector pCV-cIGF1R. pCV-cIGF1Rwas linearized with Pvu I (Takara Shuzo), extracted with phenol andchloroform, and purified by ethanol precipitation.

The linearized expression vector was introduced into mouse Ba/F3 cellsusing an electroporation apparatus (Gene Pulser: Bio Rad). The Ba/F3cells were washed twice with Dulbecco's PBS (hereinafter referred to asPBS), and then were suspended in PBS to give a cell density of about1×10⁷ cells/mL. 20 μg linearized expression vector DNA was added to 0.8mL of this suspension, transferred into a cuvette (Bio Rad) forelectroporation, and pulsed with a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in 40 mL Media A, and wereseeded on four 96-well microtiter plates (flat bottom, Falcon) at 100μL/well. 100 μL/well Media A that contained 40 ng/mL human IGF-I(Genzyme) was added, and the cells were cultured in a CO₂ incubator (CO₂concentration, 5%). RPMI1640 (GIBCO) supplemented with 10% fetal bovineserum (Hyclone), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO)was used as Media A. About one week after the start of the culture, thecells were observed under a microscope, cells were collected from wellsof a single colony and subcultured in Media A containing 20 to 50 ng/mLhuman IGF-I.

The cells were washed twice with media without human insulin(hereinafter referred to as Media B), and were suspended in Media B togive a cell density of 5×104 cells/mL. CHO-S-SFM II medium (GIBCO)without the addition of human insulin was used as Media B. 50 μl/wellcell suspension and 50 μL/well human IGF-I appropriately diluted withMedia B were dispensed into the wells of a 96-well microtiter plate(flat bottom, Falcon), and were cultured for 72 hours in a CO2 incubator(CO2 concentration, 5%). After the culture, 10 μL/well WST-8reagent(Cell Count Reagent SF; Nacalai Tesque was added, and pre-reactionabsorbance at a measurement wavelength of 450 nm and a controlwavelength of 655 nm was measured using microplate reader (Model 3550,Bio Rad). The plate was incubated for two hours in a CO2incubator (CO2concentration, 5%), and post-reaction absorbance was measured as above.Based on the cell growth activity determined by the number of viablecells by plotting the amount of changes in absorbance measured after thetwo-hour incubation on the vertical axis and the concentrations of humanIGF-I on the horizontal axis, cell line IGG#06 that had a highsensitivity to human IGF-I was selected and used as chimeric IGF-Ireceptor-expressing cell line IGG. This cell line was subcultured in thepresence of 50 ng/mL human IGF-I. At a concentration of 10 ng/mL ormore, an obvious growth reaction to human IGF-I was observed (FIG. 14).

(1-13) Leukemia Inhibitory Factor (Hereinafter Referred to as LIF)Receptor

Chimeric receptor DNAs were constructed by linking cDNA fragmentsencoding human LIF receptor (from the 1st to the 833rd amino acidresidues; Gearing, D P. et al. (1988) EMBO J., 10, 2839-2848) and humangp130 (from the 1st to the 619th amino acid residues; Hibi, M. et al.(1990) Cell, 63, 1149-1157) with the transmembrane and intracellulardomains (from the 602nd to the 813th amino acid residues; Fukunaga, R.et al. (1990) Cell 61, 341-350) of mouse G-CSF receptor, respectively.These constructs were inserted downstream of the HEF1α promoter of themammalian expression vector pCV to construct chimeric receptorsexpression vectors, pCV-cLIFR and pCV-cgp130, respectively. pCV-cLIFRand pCV-cgp130 were linearized with Pvu I (Takara Shuzo), respectively,extracted with phenol and chloroform, and purified by ethanolprecipitation.

The linearized expression vectors were introduced into mouse Ba/F3 cellsusing an electroporation apparatus (Gene Pulser: Bio Rad). The Ba/F3cells were washed twice with Dulbecco's PBS (hereinafter referred to asPBS), and then were suspended in PBS to give a cell density of about1×10⁷ cells/mL. 20 μg each of the linearized expression vector DNA,pCV-cLIFR and pCV-cgp130, was added to 0.8 mL of this suspension,transferred into a cuvette (Bio Rad) for electroporation, and pulsedwith a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in 40 mL of Media A, and wereseeded on four 96-well microtiter plates (flat bottom, Falcon) at 100μL/well. 100 μL/well Media A that contained 10 ng/mL human LIF (Genzyme)was added, and the cells were cultured in a CO₂ incubator (CO₂concentration, 5%). RPMI1640 (GIBCO) supplemented with 10% fetal bovineserum (Hyclone), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO)was used as Media A. About one week after the start of the culture, thecells were observed under a microscope, cells were collected from wellswith a single colony and subcultured in Media A containing 5 ng/mL humanLIF.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well cell suspension and50 μL/well human LIF or CNTF, each appropriately diluted in Media A,were dispensed into the wells of a 96-well microtiter plate (flatbottom, Falcon), and were cultured for 72 hours in a CO₂ incubator (CO₂concentration, 5%). After the culture, 10 μL/well WST-8 reagent (CellCount Reagent SF; Nacalai Tesque) was added, and pre-reaction absorbanceat a measurement wavelength of 450 nm and a control wavelength of 655 nmwas measured using microplate reader (Model 3550, Bio Rad). The platewas incubated for two hours in a CO₂ incubator (CO₂ concentration, 5%),and post-reaction absorbance was measured as above. Based on the cellgrowth activity determined by the number of viable cells by plotting theamount of changes in absorbance measured after the two-hour incubationon the vertical axis and the concentrations of human LIF on thehorizontal axis, cell line LIG#47 that had a high sensitivity to humanLIF was selected and used as chimeric LIF receptor-expressing cell lineLIG. This cell line was subcultured in the presence of 1 ng/mL humanLIF. At a concentration of 0.4 ng/mL or more, an obvious growth reactionto human LIF could be observed (FIG. 15).

(1-14) Ciliary Neurotrophic Factor (Hereinafter Referred to as CNTF)Receptor

Human CNTF receptor (from the 1st to the 372nd amino acid residues;Davis, S. et al. (1991) Science, 253, 59-63) cDNA was cloned downstreamof the HEF1α promoter of a mammalian expression vector, pCV, toconstruct human CNTF receptor (CNTFR) expression vector, pCV-CNTFR.pCV-CNTFR was linearized with Pvu I (Takara Shuzo), extracted withphenol and chloroform, and purified by ethanol precipitation.

The linearized expression gene vector was introduced into the chimericLIF receptor-expressing cell line LIG (see section 1-13) using anelectroporation apparatus (Gene Pulser: Bio Rad). The LIG cells werewashed twice with Dulbecco's PBS (hereinafter referred to as PBS), andthen were suspended in PBS to give a cell density of about 1×10⁷cells/mL. 10 μg of linearized pCV-CNTFR was added to 0.8 mL of thissuspension, transferred into a cuvette (Bio Rad) for electroporation,and pulsed with a capacitance of 960 μF at 0.33 kV.

After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in 40 mL of Media A, and wereseeded on four 96-well microtiter plates (flat bottom, Falcon) at 100μL/well. 100 μL/well Media A that contained 1 ng/mL human CNTF (Genzyme)was added, and the cells were cultured in a CO₂ incubator (CO₂concentration, 5%). RPMI1640 (GIBCO) supplemented with 10% fetal bovineserum (Hyclone), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (GIBCO)was used as Media A. About one week after the start of the culture, thecells were observed under a microscope, cells were collected from wellswith a single colony and subcultured in Media A containing 0.5 to 1ng/mL CNTF.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well cell suspension and50 μL/well human CNTF appropriately diluted in Media A were dispensedinto the wells of a 96-well microtiter plate (flat bottom, Falcon), andwere cultured for 72 hours in a CO₂ incubator (CO₂ concentration, 5%).After the culture, 10 μL/well WST-8 reagent (Cell Count Reagent SF;Nacalai Tesque) was added, and pre-reaction absorbance at a measurementwavelength of 450 nm and a control wavelength of 655 nm was measuredusing microplate reader (Model 3550, Bio Rad). The plate was incubatedfor two hours in a CO₂ incubator (CO₂ concentration, 5%), andpost-reaction absorbance was measured as above. Based on the cell growthactivity determined by the number of viable cells by plotting the amountof changes in absorbance measured after the two-hour incubation on thevertical axis and the concentrations of human CNTF on the horizontalaxis, cell line CNG#203 that had a high sensitivity to human CNTF wasselected and used as chimeric CNTF receptor-expressing cell line CNG.This cell line was subcultured in the presence of 1 ng/mL human CNTF. Ata concentration of 0.3 ng/mL or more, an obvious growth reaction tohuman CNTF was observed (FIG. 16).

CNTF receptor has been known to form a heterotrimer, not a homodimer, totransmit signal. According to the present experiment, a chimericreceptor of such a heterotrimeric receptor, even if the intracellulardomain of the receptor was replaced with that of a homodimeric receptor(represented by the G-CSF receptor), could induce signal transductionand the reactivity of the ligand could be detected as the cell growthactivity.

(1-15) Platelet-Derived Growth Factor (Hereinafter Referred to as PDGF)Receptor

A chimeric receptor cDNA was constructed by linking cDNA fragmentsencoding the β chain (from the 1st to the 531st amino acid residues;Gronwald, R G K. et al. (1988) Proc. Natl. Acad. Sci. USA. 85,3435-3439) of human PDGF receptor and the transmembrane andintracellular domains (from the 602nd to the 813th amino acid residues;Fukunaga, R. et al. (1990) Cell 61, 341-350) of mouse G-CSF receptor.The cDNA was cloned downstream of the EF1α promoter of the mammalianexpression vector pCV to construct chimeric receptor-expression vectorpCV-cPDGFR. pCV-cPDGFR was linearized with Pvu I (Takara Shuzo),extracted with phenol and chloroform, and purified by ethanolprecipitation.

The linearized expression vector was introduced into mouse Ba/F3 cellsusing an electroporation apparatus (Gene Pulser: Bio Rad). The Ba/F3cells were washed twice with Dulbecco's PBS (hereinafter referred to asPBS), and then were suspended in PBS to give a cell density of about1×10⁷ cells/mL. 20 μg of the linearized expression vector DNA was addedto 0.8 mL of this suspension, transferred into a cuvette (Bio Rad) forelectroporation, and pulsed with a capacitance of 960 μF at 0.33 kV.After leaving standing for about 10 min at room temperature, the cellstreated by electroporation were suspended in Media A, and were seeded ona 96-well microtiter plate (flat bottom, Falcon) at 100 μL/well. 100μL/well Media A that contained 20 ng/mL human PDGF-BB (Genzyme) wasadded, and the cells were cultured in a CO₂ incubator (CO₂concentration, 5%). RPMI1640 (GIBCO) supplemented with 10 vol % fetalbovine serum (Hyclone), 100 U/mL penicillin, and 0.1 mg/mL streptomycin(GIBCO) was used as Media A. About one week after the start of theculture, the cells were observed under a microscope, cells werecollected from wells with a single colony and subcultured in Media Acontaining 5 ng/mL human PDGF-BB.

The cells were washed twice with Media A, and were suspended in Media Ato give a cell density of 5×10⁴ cells/mL. 50 μl/well cell suspension and50 μL/well human PDGF-BB appropriately diluted in Media A were dispensedinto the wells of a 96-well microtiter plate (flat bottom, Falcon), andwere cultured for 72 hours in a CO₂ incubator (CO₂ concentration, 5%).After the culture, 10 μL/well WST-8 reagent (Cell Count Reagent SF;Nacalai Tesque) was added, and pre-reaction absorbance at a measurementwavelength of 450 nm and a control wavelength of 655 nm was measuredusing microplate reader (Model 3550, Bio Rad). The plate was incubatedfor four hours in a CO₂ incubator (CO₂ concentration, 5%), andpost-reaction absorbance was measured as above. Based on the cell growthactivity determined by the number of viable cells by plotting the amountof changes in absorbance measured after the 4-hour incubation on thevertical axis and the concentrations of human PDGF-BB on the horizontalaxis, cell line PDG#10 that had a high sensitivity to human PDGF-BB wasselected and used as chimeric PDGF receptor-expressing cell line PDG(FIG. 17).

EXAMPLE 2 Examination on Cytokine Responses in Mixed Cell Cultures

Mixed culture of the cell lines GFG, EPG, and TPG, which express humanG-CSF, EPO, and TPO chimeric receptors, respectively, and their parentalcell line Ba/F3 was conducted and their response to various cytokineswas examined.

Four cell lines, EPG, GFG, TPG, and the non-recombinant cell Ba/F3, weresubcultured in the presence of 1 ng/mL human EPO, 10 ng/mL human G-CSF,1 ng/mL human TPO (R & D Systems), and 1 ng/mL mouse IL-3 (R & DSystems), respectively. The used human EPO and G-CSF were prepared fromrecombinant CHO cells, and their titer was 270,000 IU/mg and 1.2×10⁸IU/mg, respectively. The cells were washed twice with RPMI1640 mediacontaining 2% fetal bovine serum, and then were suspended in RPMI1640containing 10% fetal bovine serum. The four cell lines were mixed byadjusting each of the cell lines to 4×10⁴ cells/mL (16×10⁴ cells/mL intotal) for the mixed culture, and, for individual cultures, cells wereprepared at 4×10⁴ cells/mL. 100 μL each of the cells were independentlyseeded on a 96-well microtiter plate (flat bottom, Falcon). After adding100 μL/well each of appropriately diluted cytokines, the cultures werecultured for 72 hours in a CO₂ incubator (5% CO₂ concentration). Afterthe culture, 10 μL/well WST-8 reagent (Cell Count Reagent SF; NacalaiTesque) was added, and pre-reaction absorbance at a measurementwavelength of 450 nm and a control wavelength of 655 nm was measuredusing a microplate reader (Model 3550, Bio Rad). The plate was incubatedfor two hours in a CO₂ incubator (CO₂ concentration, 5%), andpost-reaction absorbance was measured as above. Data were graphed outtaking the cytokine concentrations on the horizontal axis and the amountof changes in absorbance after 2-hour incubation on the vertical axis(FIGS. 18, 19, and 20).

The responses of the mixed culture against cytokines began at similarconcentration (2 pg/mL of hEPO, 200 pg/mL of hTPO, and 2 pg/mL ofhG-CSF) and a similar growth activity was detected as in the individualcultures. The growth of the chimeric receptor-expressing cells wasspecifically induced by corresponding ligands. The parental cell linedid not respond to any cytokine, and no growth activity was observed.

INDUSTRIAL APPLICABILITY

According to the screening methods of the present invention, two or moredifferent activities can be simultaneously assayed with the same index.Therefore, the methods of the present invention enables an efficient andrapid screening for a vast number of test samples to isolate substanceshaving biological activities of interest. The methods of the presentinvention are particularly suited for the screening of ligands that bindto certain receptors, and serves as an important basic technique for thedevelopment of novel pharmaceuticals.

1. A method of screening for a ligand that can bind to at least one oftwo or more kinds of receptors, wherein said method comprises the stepsof: (i)providing a composition comprising cells transformed withexpression vectors encoding two or more kinds of receptors andexpressing said two or more kinds of receptors, each of said receptorscomprising (a) a common signal-transducing domain derived from areceptor selected from the group consisting of hematopoietic factorreceptor family, cytokine receptor family, tyrosine kinase-type receptorfamily, serine/threonine kinase-type receptor family, TNF receptorfamily, G protein-coupled receptor family, GPI-anchored receptor family,tyrosine phosphatase-type receptor family, cell adhesion receptorfamily, and hormone receptor family, and (b) a ligand-binding domainderived from a receptor selected from the group consisting ofhematopoietic factor receptor family, cytokine receptor family, tyrosinekinase-type receptor family, serine/threonine kinase-type receptorfamily, TNF receptor family, G protein-coupled receptor family,GPI-anchored receptor family, tyrosine phosphatase-type receptor family,cell adhesion receptor family, and hormone receptor family, wherein ineach of said kinds of receptors, said ligand-binding domain derives froma different receptor, and binding of a ligand to the ligand-bindingdomain induces signal transduction through the signal-transducingdomain; (ii) contacting a test sample with said composition comprisingcells expressing two or more kinds of receptors; and (iii) detectingbinding of said test sample to at least one of said two or more kinds ofreceptors by detecting a change in a detection marker selected from thegroup consisting of proliferation activity of the cells, phosphorylationof the receptor or downstream substrate proteins, dephosphorylation ofthe receptor or downstream substrate proteins, change in cAMP level,change in Ca²⁺ level, and induction of downstream gene expression,wherein said change is induced by signal transduction through saidcommon signal-transducing domain.
 2. The method according to claim 1,wherein the cells in the composition comprise two or more kinds ofcells, each kind of cell expressing at least one of said two or morekinds of receptors.
 3. The method according to claim 1, wherein saidsignal-transducing domain is derived from a receptor selected from thegroup consisting of human or mouse erythropoietin (EPO) receptor, humanor mouse granulocyte-colony stimulating factor (G-CSF) receptor, humanor mouse thrombopoietin (TPO) receptor, and human or mouse epidermalgrowth factor (EGF) receptor, and wherein said ligand-binding domain isderived from a receptor selected from the group consisting of human ormouse EPO receptor, human or mouse G-CSF receptor, human or mouse TPOreceptor, human or mouse insulin receptor, human or mouse Flt-3receptor, human or mouse platelet-derived growth factor (PDGF) receptor,human or mouse interferon (IFN)-α or -β receptor, human or mouse leptinreceptor, human or mouse growth hormone (GH) receptor, human or mouseinterleukin (IL)-10 receptor, human or mouse insulin-like growth factor(IGF)-I receptor, human or mouse leukemia inhibitory factor (LIF)receptor, and human or mouse ciliary neurotrophic factor (CNTF)receptor.
 4. The method according to claim 3, wherein saidsignal-transducing domain derives from mouse G-CSF receptor.
 5. Themethod according to claim 1, wherein said cells are derived from acytokine-dependent cell.
 6. The method according to claim 5, whereinsaid cells are derived from a Ba/F3 cell or a FDC-P1 cell.
 7. The methodaccording to claim 1, which method further comprises the step ofcontacting the test sample with one of said two or more receptorsprovided in (i) in order to determine the specificity of binding of thetest sample.
 8. The method of claim 1, wherein, in at least two of saidtwo or more kinds of receptors, said ligand-binding domain derives froma receptor different from the receptor from which the commonsignal-transducing domain of (i)(a) is derived.
 9. A method ofidentifying a ligand that can bind to at least one of two or more kindsof receptors, wherein said method comprises the steps of: (i) providinga composition comprising cells expressing two or more kinds of chimericreceptors, each of said chimeric receptors comprising: (a) a commonsignal-transducing domain derived from a receptor selected from thegroup consisting of hematopoietic factor receptor family, cytokinereceptor family, tyrosine kinase-type receptor family, serine/threoninekinase-type receptor family, TNF receptor family, G protein-coupledreceptor family, GPI-anchored receptor family, tyrosine phosphatase-typereceptor family, cell adhesion receptor family, and hormone receptorfamily, and (b) an extracellular domain or ligand-binding portionthereof derived from a receptor selected from the group consisting ofhematopoietic factor receptor family, cytokine receptor family, tyrosinekinase-type receptor family, serine/threonine kinase-type receptorfamily, TNF receptor family, G protein-coupled receptor family,GPI-anchored receptor family, tyrosine phosphatase-type receptor family,cell adhesion receptor family, and hormone receptor family, wherein, ineach kind of chimeric receptor, said extracellular domain orligand-binding portion thereof derives from a different receptor, andbinding of a ligand to the extracellular domain or ligand-bindingportion thereof induces signal transduction through thesignal-transducing domain, (ii) contacting said composition with a testsample, and (iii) detecting binding of said test sample to at least oneof said two or more kinds of receptors by detecting a change in adetection marker selected from the group consisting of proliferationactivity of the cells, phosphorylation of the receptor or downstreamsubstrate proteins, dephosphorylation of the receptor or downstreamsubstrate proteins, change in cAMP level, change in Ca²⁺ level, andinduction of downstream gene expression, wherein said change is inducedby signal transduction through said common signal-transducing domain.10. The method of claim 1, wherein said common signal-transducing domainis derived from a receptor selected from the group consisting of EPOreceptor, G-CSF receptor, TPO receptor, and EGF receptor.
 11. The methodof claim 1, wherein the ligand-binding domain of each of said two ormore kinds of receptors is derived from a receptor selected from thegroup consisting of EPO receptor, G-CSF receptor, TPO receptor, insulinreceptor, Flt-3 receptor, PDGF receptor, interferon (IFN)-α orβ-receptor, leptin receptor, GH receptor, IL-10 receptor, IGF-Ireceptor, LIF receptor, and CNTF receptor.
 12. The method of claim 1,wherein in step (iii), detecting of binding of said test sample to atleast one of said two or more kinds of receptors is by detecting achange in proliferation activity of the cells, wherein said change isinduced by signal transduction through said common signal-transducingdomain.
 13. A method of screening for a ligand that can bind to at leastone of two or more kinds of receptors, wherein said method comprises thesteps of: (i) providing a composition comprising cells expressing two ormore kinds of receptors, each of said receptors comprising (a) a commonsignal-transducing domain derived from a receptor selected from thegroup consisting of EPO receptor, G-CSF receptor, TPO receptor, and EGFreceptor, and (b) a ligand-binding domain derived from a receptorselected from the group consisting of EPO receptor, G-CSF receptor, TPOreceptor, insulin receptor, Flt-3 receptor, PDGF receptor, IFN-α or -βreceptor, GH receptor, IL-10 receptor, IGF-I receptor, LIF receptor, andCNTF receptor, wherein in each of said kinds of receptors, saidligand-binding domain derives from a different receptor, and binding ofa ligand to the ligand-binding domain induces signal transductionthrough the signal-transducing domain; (ii) contacting a test samplewith said composition comprising cells expressing two or more kinds ofreceptors; and (iii) detecting binding of said test sample to at leastone of said two or more kinds of receptors by detecting a change inproliferation activity of the cells, wherein said change is induced bysignal transduction through said common signal-transducing domain.
 14. Amethod of identifying a ligand that can bind to at least one of two ormore kinds of receptors, wherein said method comprises the steps of: (i)providing a composition comprising cells expressing two or more kinds ofchimeric receptors, each of said chimeric receptors comprising: (a) acommon signal-transducing domain derived from a receptor selected fromthe group consisting of EPO receptor, G-CSF receptor, TPO receptor, andEGF receptor, and (b) an extracellular domain or ligand-binding portionthereof derived from a receptor selected from the group consisting ofEPO receptor, G-CSF receptor, TPO receptor, insulin receptor, Flt-3receptor, PDGF receptor, IFN-α or β-receptor, leptin receptor, GHreceptor, IL-10 receptor, IGF-I receptor, LIF receptor, and CNTFreceptor, wherein, in each kind of chimeric receptor, said extracellulardomain or ligand-binding portion thereof derives from a differentreceptor, and binding of a ligand to the extracellular domain orligand-binding portion thereof induces signal transduction through thesignal-transducing domain, (ii) contacting said composition with a testsample, and (iii) detecting binding of said test sample to at least oneof said two or more kinds of receptors by detecting a change inproliferation activity of the cells, wherein said change is induced bysignal transduction through said common signal-transducing domain.